Glycoproteins having lipid mobilizing properties and therapeutic uses thereof

ABSTRACT

The invention provides formulations and methods for ameliorating symptoms associated with metabolic disorders, such as cachexia, hypoglycemia, obesity, diabetes, and the like by administering Zn-α 2 -glycoproteins or a functional fragment thereof, alone or in combination with additional agents, such as β adrenergin receptor agonists, β adrenergin receptor antagonists, and/or glycemic control agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/805,190, filed Feb. 20, 2013, currently pending; which is a 35 USC§371 National Stage application of International Application No.PCT/GB2011/000966 filed Jun. 27, 2011, now pending; which claims thebenefit under 35 USC §119(e) to U.S. Application Ser. No. 61/420,677filed Dec. 7, 2010, to U.S. Application Ser. No. 61/384,652 filed Sep.20, 2010 and to U.S. Application Ser. No. 61/358,596 filed Jun. 25,2010, all now expired. The disclosure of each of the prior applicationsis considered part of and is incorporated by reference in the disclosureof this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to medicinal formulations andsupplements, and more particularly, to formulations and methods foraltering the metabolism of a subject, as well as ameliorating disorderssuch as cachexia, obesity, diabetes and insulin resistance.

2. Background Information

The prevalence of obesity in adults, children and adolescents hasincreased rapidly over the past 30 years in the United States andglobally and continues to rise. Obesity is classically defined based onthe percentage of body fat or, more recently, the body mass index (BMI),also called Quetlet index (National Task Force on the Prevention andTreatment of Obesity, Arch. Intern. Med., 160: 898-904 (2000);Khaodhiar, L. et al., Clin. Cornerstone, 2: 17-31 (1999)). The BMI isdefined as the ratio of weight (kg) divided by height (in meters)squared.

Overweight and obesity are associated with increasing the risk ofdeveloping many chronic diseases of aging seen in the U.S. Suchco-morbidities include type 2 diabetes mellitus, hypertension, coronaryheart diseases and dyslipidemia, gallstones and cholecystectomy,osteoarthritis, cancer (of the breast, colon, endometrial, prostate, andgallbladder), and sleep apnea. It is estimated that there are around325,000 deaths annually that are attributable to obesity. The key toreducing the severity of the diseases is to lose weight effectively.Although about 30 to 40% claim to be trying to lose weight or maintainlost weight, current therapies appear not to be working. Besides dietarymanipulation, pharmacological management and in extreme cases, surgery,are sanctioned adjunctive therapies to treat overweight and obesepatients (Expert Panel, National Institute of Health, Heart, Lung, andBlood Institute, 1-42 (June 1998); Bray, G. A., Contemporary Diagnosisand Management of Obesity, 246-273 (1998)). Drugs have side effects, andsurgery, although effective, is a drastic measure and reserved formorbidly obese.

Cachexia is wasting of both adipose and skeletal muscle mass caused bydisease. It occurs in many conditions and is common with many cancerswhen remission or control fails. Patients with advanced cancer, AIDS,and some other major chronic progressive diseases may appear cachectic.Cachexia can occur in people who are eating enough, but who cannotabsorb the nutrients. While cachexia may be mediated by certaincytokines, especially tumor necrosis factor-α, IL-1b, and IL-6, whichare produced by tumor cells and host cells in the tissue mass, there iscurrently no widely accepted treatment for cachexia.

Diabetes mellitus is a major cause of morbidity and mortality.Chronically elevated blood glucose leads to debilitating complicationsnephropathy, often necessitating dialysis or renal transplant;peripheral neuropathy; retinopathy leading to blindness; ulceration ofthe legs and feet, leading to amputation; fatty liver disease, sometimesprogressing to cirrhosis; and vulnerability to coronary artery diseaseand myocardial infarction.

There are two primary types of diabetes. Type I, or insulin-dependentdiabetes mellitus (IDDM) is due to autoimmune destruction ofinsulin-producing beta cells in the pancreatic islets. The onset of thisdisease is usually in childhood or adolescence. Treatment consistsprimarily of multiple daily injections of insulin, combined withfrequent testing of blood glucose levels to guide adjustment of insulindoses, because excess insulin can cause hypoglycemia and consequentimpairment of brain and other functions. Increasing scrutiny is beinggiven to the role of insulin resistance to the genesis, progression, andtherapeutic management of this type of diabetic disease.

Type II, or noninsulin-dependent diabetes mellitus (NIDDM) typicallydevelops in adulthood. NIDDM is associated with resistance ofglucose-utilizing tissues like adipose tissue, muscle, and liver, to theactions of insulin. Initially, the pancreatic islet beta cellscompensate by secreting excess insulin. Eventual islet failure resultsin decompensation and chronic hyperglycemia. Conversely, moderate isletinsufficiency can precede or coincide with peripheral insulinresistance. There are several classes of drugs that are useful fortreatment of NIDDM 1) insulin releasers, which directly stimulateinsulin release, carrying the risk of hypoglycemia; 2) prandial insulinreleasers, which potentiate glucose-induced insulin secretion, and mustbe taken before each meal; 3) biguanides, including metformin, whichattenuate hepatic gluconeogenesis (which is paradoxically elevated indiabetes); 4) insulin sensitizers, for example the thiazolidinedionederivatives rosiglitazone and pioglitazone, which improve peripheralresponsiveness to insulin, but which have side effects like weight gain,edema, and occasional liver toxicity; 5) insulin injections, which areoften necessary in the later stages of NIDDM when the islets have failedunder chronic hyperstimulation.

Insulin resistance can also occur without marked hyperglycemia, and isgenerally associated with atherosclerosis, obesity, hyperlipidemia, andessential hypertension. This cluster of abnormalities constitutes the“metabolic syndrome” or “insulin resistance syndrome”. Insulinresistance is also associated with fatty liver, which can progress tochronic inflammation (NASH; “nonalcoholic steatohepatitis”), fibrosis,and cirrhosis. Cumulatively, insulin resistance syndromes, including butnot limited to diabetes, underlie many of the major causes of morbidityand death of people over age 40.

Despite the existence of such drugs, diabetes remains a major andgrowing public health problem. Late stage complications of diabetesconsume a large proportion of national health care resources. There is aneed for new orally active therapeutic agents which effectively addressthe primary defects of insulin resistance and islet failure with feweror milder side effects than existing drugs.

Zinc-α₂-glycoprotein (ZAG) has been identified as a lipid mobilizingfactor (LMF) with the potential to induce fat loss in cancer cacehxia.ZAG was shown to induce lipolysis in white adipocytes by interactionwith a β3-adrenergic receptor, while in vivo it increased expression ofuncoupling protein-1 (UCP-1) in brown adipose tissue (BAT), and inducedloss of body fat. In addition to some tumors, ZAG is also produced bywhite adipose tissue (WAT) and BAT and its expression is upregulated incachexia. In contrast ZAG expression in adipose tissue of obese humanswas only 30% of that found in non-obese subjects. This suggests thatloss of ZAG expression in WAT could account for some of the features ofobesity. Certainly inactivation of both ZAG alleles in mice led to anincrease in body weight which was more pronounced when the animals werefed a high fat diet. The lipolytic response to various agents wassignificantly decreased in adipocytes from ZAG deficient animals.

To date studies on the lipid mobilizing effect of ZAG have been carriedout in both mice and rats using human and murine ZAG. The studiesindicate that ZAG is evolutionarily conserved and exhibits cross-speciesactivity, e.g., murine ZAG exhibiting substantially the same activity inhumans and vice-versa.

There remains a lack of effective and safe alternatives for alteringmetabolism and treatment of metabolic diseases, such as obesity,diabetes and cachexia. There is therefore a need for new formulationsfor such uses.

SUMMARY OF THE INVENTION

The present invention is based in part on the finding thatZinc-α₂-glycoprotein has an effect on body weight and insulinresponsiveness in adult obese hyperglycemic (ob/ob) mice and matureWistar rats, and that anti-ZAG antibodies prevent weight loss incachexia situations. Such a finding is useful in methods for moderatingbody weight, improving insulin responsiveness or ameliorating thesymptoms associated with cachexia or diseases associated with musclewasting.

In one embodiment the present invention provides a formulationcomprising a zinc-α₂-glycoprotein (ZAG), a ZAG variant, a modified ZAG,or a functional fragment thereof. In one aspect; the ZAG is mammalian,e.g., human, and may include the amino acid sequence set forth in SEQ IDNO: 1. The ZAG peptide may conjugated to a non-protein polymer. The ZAGpeptide may be sialylated, PEGylated or modified to increase solubilityor stability. The ZAG peptide may be recombinant or synthetic. Invarious aspects, the ZAG peptide may be modified ZAG and include thewild-type ZAG amino acid sequence with one or more mutations to theamino acid sequence selected from deletions, additions or conservativesubstitutions. In various aspects, the ZAG peptide may include one ormore of a leader sequence and a trailing sequence. The ZAG peptide mayalso be glycosylated, e.g., as a result of a posttranslationalmodification. Additionally, in various embodiments the formulation mayfurther include a pharmaceutically acceptable carrier. The formulationmay also include one or more agents including a β3 agonist andβ-adrenergic receptor (β-AR) antagonist, such as a β2-adrenergicreceptor (β2-AR) antagonist, a β1-adrenergic receptor (β1-AR)antagonist, and a β3-adrenergic receptor (β3-AR) antagonist. In someaspects, the formulation of claim 1 may further include a glucagon-likepeptide-1 (GLP-1) or an analog thereof.

In another embodiment, the invention provides a foodstuff additive ornutritional supplement including the formulation of the invention asdescribed herein.

In another embodiment, the invention provides a method for delivering aformulation to a mammalian subject, the method including administeringto the mammalian subject the formulation as described herein.

In another embodiment, the invention provides a method for delivering azinc-α₂-glycoprotein (ZAG) to a mammalian subject, the method includingdelivering to the subject by oral administration the formulation asdescribed herein.

In another embodiment, the invention provides a method for orallydelivering a zinc-α₂-glycoprotein (ZAG) to a mammalian subject in megadoses similar to that of mega dosed oral insulin requiring systemicabsorption of administered ZAG as described herein.

In another embodiment, the invention provides a method for orallydelivering a zinc-α₂-glycoprotein (ZAG) to a mammalian subject insurprisingly effective low doses similar to that of intravenousadministration of ZAG and in formulations surprisingly not requiringsystemic absorption of administered ZAG as described herein.

In another embodiment, the invention provides a method for increasing asubject's endogenous level of a zinc-α₂-glycoprotein (ZAG), the methodincluding administering to the subject the formulation as describedherein.

The present invention further provides a method of ameliorating symptomsof cachexia in a subject. The method includes administering to thesubject in need of such treatment a therapeutically effective dosage ofan inhibitor of the biological activity of a polypeptide having thesequence as shown in SEQ ID NO: 1, resulting in an amelioration ofsymptoms associated with cachexia following treatment. In oneembodiment, the inhibitor is a monoclonal antibody that binds apolypeptide that comprises a sequence at least 80% homologous to thepolypeptide having the sequence as shown in SEQ ID NO: 1. In anotherembodiment, the treatment includes daily administration for 10 days. Inanother embodiment, the inhibitor is administered daily, every otherday, every 2 days, or every 3 days, for up to 10 days or longer. Inanother embodiment, the antibody is administered twice daily. Theantibody may be administered intravenously, subcutaneously,sublingually, intranasally, orally, or via inhalation. In anotherembodiment, the inhibitor is administered in combination with one ormore agents selected from the group consisting of a β3-adrenergicreceptor (β3-AR) antagonist. In one embodiment, the β3-AR antagonist isSR59230A. In another embodiment, the antibody is glycosylated. Inanother embodiment, the agent that inhibits the homologous polypeptideis a non-antibody agent, for example but not limited to, an aptamer.

In another aspect, the present invention provides a method of treating asubject to bring about reduction in weight loss. The method includesadministering to the subject in need of such treatment a therapeuticallyeffective dosage of an inhibitor of the polypeptide having the sequenceas shown in SEQ ID NO: 1 in combination with one or more agents selectedfrom the group consisting of a β3-adrenergic receptor (β3-AR)antagonist. In one embodiment, the inhibitor is a monoclonal antibodythat binds a polypeptide that comprises a sequence at least 80%homologous to the polypeptide having the sequence as shown in SEQ IDNO: 1. In another embodiment, the β3-AR antagonist is SR59230A. Inanother embodiment, the antibody is glycosylated. In another embodiment,the agent that inhibits the homologous polypeptide is a non-antibodyagent, for example but not limited to, an aptamer.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an antibody, or functional fragment thereof, thatbinds the polypeptide having the sequence as shown in SEQ ID NO: 1 andan agent selected from the group consisting of a β3-adrenergic receptor(β3-AR) antagonist and a β3 antagonist. In one embodiment, the β3-ARantagonist is SR59230A. In another embodiment, the antibody isglycosylated.

This disclosure provides materials and methods for supplementing a humanor animal diet. In one aspect, the instant disclosure providesnutritional supplement formulations. A nutritional supplementformulation of the invention can include zinc-α2-glycoprotein (ZAG) or afunctional fragment thereof. The disclosure also provides materials suchas kits that include one or more nutritional supplement formulations,such as nutritional supplement formulations that include ZAG orfunctional fragments thereof. In another aspect, the disclosure providesa method of delivery of an orally administered therapeutic agent,including administering a β3 agonist in combination with the orallyadministered therapeutic agent.

The formulations, kits, and methods herein can be useful for improving ahuman's health and/or to promote weight loss, or independent of weightloss, improve insulin resistance and reduce hyperglycemia. Theseformulations, kits and methods may therefore find use in the treatmentof diseases associated with obesity and/or, hyperglycemia.

In another aspect, the invention provides a food stuff that includes theformulation of the invention in combination with a consumable carrier.Exemplary consumable carriers include, but are not limited to cookies,brownies, crackers, breakfast bars, energy bars, cereals, cakes, breads,beverages, meat products, and meat substitute products.

In another aspect, the invention provides a method of supplementing ahuman diet. The method includes ingesting formulation that includeszinc-α₂-glycoprotein (ZAG) or a functional fragment thereof. In oneembodiment, the ZAG is mammalian, such as the human ZAG polypeptidehaving the sequence as shown in SEQ ID NO: 1, or a fragment thereof. Themethod may be performed daily for 10 days. In another embodiment, theformulation is ingested daily, every other day, every 2 days, or every 3days, for up to 10 days or longer. In another embodiment, theformulation is ingested twice daily. In another embodiment, theformulation is ingested in combination with one or more agents selectedfrom the group consisting of a β3-adrenergic receptor (β3-AR) agonistand a βAR agonist and a β3-AR antagonist. In one embodiment, the β3-ARantagonist is SR59230A. In another embodiment, β3-AR agonist isAMNI-BRL37344 (BRL37344). In another embodiment, the formulation isingested or delivered in combination with one or more agents used toimprove glycemic control whether sequentially in any order or inparallel. In one embodiment, the glycemic control agent is insulin orany derivative or analog thereof. In another embodiment, the glycemiccontrol agent is a glucagon-like peptide-1 (GLP-1) or any derivative oranalog thereof.

In another aspect, the invention provides a method of delivery of anorally administered therapeutic agent, wherein the therapeutic agent isdelivered in combination with β3 agonist. In another embodiment, the β3agonist and the therapeutic agent are delivered simultaneously. In yetanother embodiment, the β3 agonist is administered prior to or followingadministration of the therapeutic agent. In certain embodiments, thetherapeutic agent is ZAG. In other embodiments, the therapeutic agentincludes atrial natriuretic peptides, brain natriuretic peptides,platelet aggregation inhibitors, streptokinase, heparin, urokinase,renin inhibitors, insulin, antibiotics, and sleep inducing peptide.

In a further aspect, the present invention provides a method of treatinga subject to bring about a weight reduction or reduction in obesity. Themethod includes administering to the subject in need of such treatment anutritional supplement formulation that includes a therapeuticallyeffective dosage of a polypeptide having the sequence as shown in SEQ IDNO: 1 or a fragment thereof.

In another embodiment, the invention provides a method of monitoringzinc-α₂-glycoprotein (ZAG) activity in a mammalian subject. The methodincludes a) orally administering the subject the formulation of theinvention; and b) detecting the level of ZAG activity; therebymonitoring ZAG activity in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a pictorial diagram showing characterization of ZAG and itseffect on lipolysis and body weight of ob/ob mice. Coomassie stainingafter 12% SDS-PAGE showing total proteins in 293 cell media and ZAGpurified as described.

FIG. 1B is a pictorial diagram showing the results of a Western blotshowing expression of ZAG in culture medium and purified ZAG.

FIG. 1C is a graphical diagram showing ZAG mRNA levels in adipose tissueand liver tissue in MAC16 mice undergoing weight loss. P<0.01.

FIG. 1D is a graphical diagram showing the results of lipolysis inepididymal adipocytes from non-obese (▪) and ob/ob mice (□) in responseto isoprenaline (Iso) and ZAG. Differences from non-obese mice are shownas *p<0.05, **p<0.01 and ***p<0.001.

FIG. 1E is a graphical diagram showing the results of lipolysis inadipocytes from epididymal (ep), subcutaneous (s.c.) and visceral (vis)deposits from obese (ob/ob) and non-obese (non ob) mice with either notreatment (▪), isoprenaline (10 μM) (□) or ZAG (0.46 μM) (

). Differences from epididymal adipocytes are shown as **p<0.01.

FIG. 1F is a graphical diagram showing the effect of ZAG (▪) on bodyweight of ob/ob mice in comparison with PBS (♦) as described in themethods. Differences in weight form time zero and PBS controls are shownas ***p<0.001.

FIG. 1G is a graphical diagram showing the effect of ZAG (□) on bodytemperature of the mice shown in e in comparison with PBS controls (▪).Differences from control are shown as ***p<0.001.

FIG. 2A is a graphical diagram showing glucose tolerance of ob/ob micetreated with ZAG. Plasma glucose levels in ob/ob mice in the fed stateeither treated with ZAG (▪) or PBS (♦) for 3 days after i.v.administration of glucose (2 g/kg). p<0.001 from PBS. Blood samples wereremoved from the tail vein at intervals after glucose administration andused for the measurement of glucose and insulin.

FIG. 2B is a graphical diagram showing plasma insulin levels in ob/obmice treated with ZAG after oral administration of glucose (1 g/kg).p<0.001 from PBS.

FIG. 2C is a graphical diagram showing glucose uptake into epididymal(ep), visceral (vis) and subcutaneous (s.c.) adipocytes of ob/ob micetreated with ZAG for 5 days in the presence of 0 (▪) 1 (□) or 10 nMinsulin (

). Differences in the presence of ZAG are indicated as ***p<0.001.

FIG. 2D is a graphical diagram showing uptake of 2-deoxy-D-glucose intogastrocnemius muscle of ob/ob mice treated with either ZAG or PBS for 5days in the absence or presence of insulin (100 nM). Differences in thepresence of insulin are shown as*p<0.05 or **p<0.01, while differencesin the presence of ZAG are shown as ***p<0.001.

FIG. 2E is a pictorial diagram showing the effect of ZAG on theexpression of GLUT4 glucose transporter in skeletal musclein of ob/obmice. After treatment of ob/ob mice for 5 days skeletal muscle wasremoved and Western blotted for expression of GLUT4.

FIG. 3A is a graphical diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed and usedfor the measurement of protein synthesis. Differences from PBS controls,or non-obese animals are shown as ***p<0.001.

FIG. 3B is a graphical diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed and usedfor the measurement of protein degradation. Differences from PBScontrols, or non-obese animals are shown as ***p<0.001.

FIG. 3C is a graphical diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed and usedfor the measurement of chymotrypsin-like enzyme activity. Differencesfrom PBS controls, or non-obese animals are shown as ***p<0.001.

FIG. 3D is a pictorial diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed andWestern blotted for expression of 20S-proteasome α-subunits.

FIG. 3E is a pictorial diagram showing the effect of ZAG on signalingpathways in skeletal muscle of ob/ob mice. After treatment of ob/ob micefor 5 days skeletal muscle was removed and Western blotted forexpression of p42.

FIG. 3F is a pictorial diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed andWestern blotted for expression of myosin.

FIG. 3G is a pictorial diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice. Aftertreatment of ob/ob mice for 5 days skeletal muscle was removed andWestern blotted for expression of actin as a control.

FIG. 4A is a pictorial diagram showing the effect of ZAG on catabolicsignaling pathways in skeletal muscle by Western blotting of phospho PKRin gastrocnemius muscle of ob/ob mice after treatment with either PBS orZAG for 5 days. The total forms of the proteins serve as loadingcontrols. Differences from PBS controls are shown as ***p<0.001 whiledifferences from non-obese mice are shown as # p<0.001.

FIG. 4B is a pictorial diagram showing the effect of ZAG on catabolicsignaling pathways in skeletal muscle by Western blotting of phosphoeIF2α in gastrocnemius muscle of ob/ob mice after treatment with eitherPBS or ZAG for 5 days. The total forms of the proteins serve as loadingcontrols. Differences from PBS controls are shown as ***p<0.001 whiledifferences from non-obese mice are shown as # p<0.001.

FIG. 4C is a pictorial diagram showing the effect of ZAG on catabolicsignaling pathways in skeletal muscle by Western blotting of phosphoPLA₂ in gastrocnemius muscle of ob/ob mice after treatment with eitherPBS or ZAG for 5 days. The total forms of the proteins serve as loadingcontrols. Differences from PBS controls are shown as ***p<0.001 whiledifferences from non-obese mice are shown as # p<0.001.

FIG. 4D is a pictorial diagram showing the effect of ZAG on catabolicsignaling pathways in skeletal muscle by Western blotting of phosphop38MAPK in gastrocnemius muscle of ob/ob mice after treatment witheither PBS or ZAG for 5 days. The total forms of the proteins serve asloading controls. Differences from PBS controls are shown as ***p<0.001while differences from non-obese mice are shown as # p<0.001.

FIG. 4E is a graphical diagram showing the effect of ZAG on catabolicsignaling pathways in skeletal muscle by activity of caspase-3 (▪) andcaspase-8 (□) in gastrocnemius muscle of ob/ob mice after treatment witheither PBS or ZAG for 5 days.

FIG. 5A is a pictorial diagram showing expression of HSL in response toZAG. Western blots show expression of phospho HSL in adipocytes ofnon-obese mice 3 h after no treatment (Con), or treatment withisoprenaline (10 μM) or ZAG (0.46 μM) alone, or in the presence ofPD98059 (25 μM) after 5 days treatment with ZAG.

FIG. 5B is a pictorial diagram showing expression of HSL byimmunoblotting in epididymal (ep) adipocytes after 5 days treatment withZAG.

FIG. 5C is a pictorial diagram showing expression of HSL byimmunoblotting in subcutaneous (sc) adipocytes after 5 days treatmentwith ZAG.

FIG. 5D is a pictorial diagram showing expression of HSL byimmunoblotting in visceral (vis) adipocytes after 5 days treatment withZAG.

FIG. 5E is a pictorial diagram showing expression of ATGL in epididymaladipocytes after 5 days treatment with ZAG.

FIG. 5F is a pictorial diagram showing expression of ATGL insubcutaneous adipocytes after 5 days treatment with ZAG.

FIG. 5G is a pictorial diagram showing expression of ATGL in visceraladipocytes after 5 days treatment with ZAG.

FIG. 5H is a pictorial diagram showing expression of pERK in epididymaladipocytes after 5 days treatment with ZAG.

FIG. 5I is a pictorial diagram showing expression of pERK insubcutaneous adipocytes after 5 days treatment with ZAG.

FIG. 5J is a pictorial diagram showing expression of pERK in visceraladipocytes after 5 days treatment with ZAG.

FIG. 5K is a graphical diagram showing the response of adipocytes fromepididymal (ep), subcutaneous (sc) and visceral (vis) deposits fromob/ob mice treated with either PBS or ZAG for 5 days to the lipolyticeffect of BRL37344. Differences from PBS controls are indicated as***p<0.01, while differences in the presence of PD98059 is shown as #p<0.001.

FIG. 6A is a pictorial diagram showing the Effect of treatment of ob/obmice for 5 days with ZAG on the expression of ZAG in WAT. Western blotshowing expression of ZAG in ep, sc, and vis adipocytes. Day 0represents the day the adipocytes were removed from the mice.

FIG. 6B is a pictorial diagram showing expression of ZAG in epididymaladipocytes that were suspended in RMPI medium as described in methods.The samples were then taken out at daily intervals and Western blottedfor ZAG expression. Day 0 represents the day the adipocytes were removedfrom the mice.

FIG. 6C is a pictorial diagram showing expression of HSL in epididymaladipocytes that were suspended in RMPI medium as described in methods.The samples were then taken out at daily intervals and Western blottedfor HSL expression. Day 0 represents the day the adipocytes were removedfrom the mice.

FIG. 6D is a pictorial diagram showing expression of UCP1 in BAT removedfrom mice. Differences from PBS treated mice are shown as ***p<0.001.

FIG. 6E is a pictorial diagram showing expression of UCP3 in BAT removedfrom mice. Differences from PBS treated mice are shown as ***p<0.001.

FIG. 6F is a pictorial diagram showing expression of UCP3 ingastrocnemius muscle removed from mice. Differences from PBS treatedmice are shown as ***p<0.001.

FIG. 7A is a graphical diagram showing weight loss of the ob/ob miceduring the 21 day study. ZAG was injected at days 1, 4, 5, 8, 13, 16,18, and 19; PBS was injected at the same time points.

FIG. 7B is a graphical diagram showing weight change (g) of the ob/obmice (weight 80-90 g) during treatment with ZAG.

FIG. 7C is a graphical diagram showing increased body temperature of theob/ob mice during the 21 day study. ZAG was injected at days 1, 4, 5, 8,13, 16, 18, and 19; PBS was injected at the same time points.

FIG. 8A is a graphical diagram showing a progressive decrease in urinaryglucose excretion during the first 5 days of treatment.

FIG. 8B is a graphical diagram showing a progressive decrease in urinaryglucose excretion during the 21 day study.

FIG. 9 is a graphical diagram showing glycerol release stimulated byisoprenaline (iso) isolated adipocytes which have been in culture up to5 days from ob mice treated with and without ZAG.

FIG. 10 is a pictorial diagram showing the complete amino acid sequence(SEQ ID NO: 1) of the human plasma Zn-α₂-glycoprotein, as published byT. Araki et al. (1988) “Complete amino acid sequence of human plasmaZn-α₂-glycoprotein and its homology to histocompatibility antigens.”

FIG. 11 is a graphical diagram showing lipolytic activity of human ZAGin isolated rat epididymal adipocytes, compared with isoprenaline (10μM) in the absence or presence of SR59230A (10 μM) or anti-ZAG antibody(1:1000) (IgG). Each value is an average of 5 separate studies.Differences from control are shown as b, p<0.01 or c, p<0.001, whiledifferences from ZAG alone are indicated as e, p<0.01 or f, p<0.001.

FIG. 12A is a graphical diagram showing the effect of daily i.v.administration of either ZAG (50 μg/100 g b.w.) in 100 μl PBS (▪) or PBSalone (♦) on body weight of male Wistar rats over a 10 day period. Theprotocol for the experiment is given in the methods section.

FIG. 12B is a graphical diagram showing the body temperature of maleWistar rats administered either ZAG (▪) or PBS (♦) as described in FIG.12A.

FIG. 12C is a graphical diagram showing the uptake of 2-deoxy-D-glucoseinto epididymal adipocytes of male Wistar rats after 10 days treatmentwith either ZAG (open box) or PBS (closed box) for 10 days, as shown inFIG. 12A, in the absence or presence of insulin (60 μU/ml).

FIG. 12D is a graphical diagram showing glucose uptake intogastrocnemius muscle and BAT of male Wistar rats after 10 days treatmentwith either ZAG or PBS, in the absence or presence of insulin (60μU/ml). Differences between ZAG and PBS treated animals are shown as a,p<0.05, b, p<0.01 or c, p<0.001, while differences in the presence ofinsulin are shown as or f, p<0.001.

FIG. 12E is a graphical diagraph showing tissue Rg in ob/ob miceadministered ZAG. c, p<0.001 from PBS.

FIGS. 13A-13C are pictorial diagrams of Western blots showing expressionof GLUT4 in BAT (FIG. 13A) and WAT (FIG. 13B) and gastrocnemius muscle(FIG. 13C) of male Wistar rats treated with either PBS or ZAG for 10days as shown in FIG. 12. Differences between ZAG and PBS treatedanimals are shown as c, p<0.001.

FIGS. 14A and 14B are pictorial diagrams of Western blots showingexpression of UCP1 and UCP3 in BAT (FIG. 14A) and WAT (FIG. 14B) of maleWistar rats treated with either PBS or ZAG for 10 days as shown in FIG.12. Differences between ZAG and PBS treated animals are shown as c,p<0.001.

FIGS. 15A and 15B are pictorial diagrams of Western blots showingexpression of ATGL (FIG. 15A) and HSL (FIG. 15B) in epididymal adiposetissue of male Wistar rats treated with either PBS or ZAG for 10 days asshown in FIG. 12. Differences between ZAG and PBS treated animals areshown as c, p<0.001.

FIGS. 16A-16C are pictorial diagrams of Western blots showing expressionof ZAG in gastrocnemius muscle (FIG. 16A), WAT (FIG. 16B) and BAT (FIG.16C). Tissues were excised from male Wistar rats treated with either PBSor ZAG for 10 days as shown in FIG. 12. Differences between ZAG and PBStreated animals are shown as c, p<0.001.

FIGS. 17A and 17B are pictorial diagrams of Western blots showingexpression of phosphorylated and total forms of pPKR (FIG. 17A) andpeIF2α (FIG. 17B) in gastrocnemius muscle of male Wistar rats treatedwith either PBS or ZAG for 10 days as shown in FIG. 12. Thedensitometric analysis is the ratio of the phosphor to total forms,expressed as a percentage of the value for rats treated with PBS.

FIGS. 18A and 18B is a graphical diagram showing phenylalanine release(FIG. 18A) and protein synthesis (FIG. 18B) in C2C12 myotubes treatedwith and without ZAG for 4 h in the presence of various concentrationsof glucose. Statistically significant c, P<0.001 from control f, P<0.001from glucose alone.

FIG. 19 is a graphical diagram showing pheylalanine release in C2C12myotubes treated with and without ZAG in the presence of variousconcentrations of glucose and with and without SR59230A. Statisticallysignificant b, P<0.01 and c, P<0.001 from control; e, P<0.05 and f,P<0.001 from glucose alone.

FIG. 20 is a graphical diagram showing protein synthesis in C2C12myotubes treated with and without ZAG in the presence of variousconcentrations of glucose and with and without SR59230A. Statisticallysignificant b, P<0.01 and c, P<0.001 from control; e, P<0.05 and f,P<0.001 from glucose alone; I, P<0.001 from glucose+SR.

FIG. 21 is a graphical diagram showing ROS activity in C2C12 myotubestreated with various concentrations of glucose with and without ZAG.Statistically significant c, P<0.001 from control f, P<0.001 fromglucose alone.

FIG. 22A is a pictorial diagram of a Western blot showing pPKR in C2C12myotubes treated with glucose with and without ZAG. Statisticallysignificant c, P<0.001 from control f, P<0.001 from glucose alone.

FIG. 22B is a pictorial diagram of a Western blot showing peIF2α inC2C12 myotubes treated with glucose with and without ZAG. Statisticallysignificant c, P<0.001 from control f, P<0.001 from glucose alone.

FIGS. 23A and 23B are graphical diagrams showing the results of aninsulin tolerance test in ob/ob mice treated with and without ZAG.Statistically significant b, P<0.05 and c, P<0.001 from with ZAG.

FIG. 24 is a graphical diagram showing the oxidation of D-[U-⁴C glucose]to ¹⁴CO₂ in ob/ob mice.

FIG. 25 is a graphical diagram showing production of ¹⁴CO₂ from [¹⁴Ccarboxy] triolein in ob/ob mice.

FIG. 26 is a graphical diagram showing reduction in weight loss in miceadministered anti-ZAG, as compared to mice administered BRL37344(cachexia model).

FIG. 27 is a graphical diagram showing glucose tolerance in ob/ob micetreated with the β3 agonist, BRL37344 in the absence and presence of ananti-ZAG antibody.

FIG. 28 is a graphical diagram showing the results of lipolysis inepididymal murine adipocytes in response to isoprenaline (Iso), ZAG, andan anti-ZAG antibody.

FIG. 29 is a graphical diagram showing weight change in ob/ob micetreated with BRL in the absence or presence of anti-ZAG where BRL wasadded either 24 h prior to anti-ZAG ab or at the same time.

FIG. 30 is a graphical diagram showing decreased proteolysis andincreased muscle synthesis in ZAG treated ob/ob mice.

FIG. 31 is a graphical diagram showing weight change in ob/ob micetreated with and without ZAG.

FIG. 32 is a graphical diagram showing body temperature in ob/ob micetreated with and without ZAG.

FIG. 33 is a graphical diagram showing urine glucose levels in ob/obmice treated with and without ZAG.

FIG. 34 is a pictorial diagram of a Western blot showing ZAG in ob/obmice following oral administration. Treatment with rhZAG administeredorally causes an increase in endogenously expressed murine ZAG inplasma.

FIG. 35 is a pictorial diagram of a Western blot showing ZAG expressionin WAT from ob/ob mice treated with and without human ZAG (p.o.).Treatment with rhZAG administered orally causes an increase inendogenously expressed murine ZAG in WAT.

FIG. 36 is a graphical diagram showing weight change in ob/ob micetreated with and without ZAG (p.o.) in the absence or presence ofpropranolol, a general β-AR antagonist. Propranolol was increased from20 to 40 mg/kg on day 3, after which change in weight loss altered fromthe negative slope of the ZAG-treated animals to the positive slope ofthe untreated animals.

FIG. 37 is a graphical diagram showing change in body temperature inob/ob mice treated with and without ZAG (p.o.) in the absence orpresence of propranolol. Propranolol was increased from 20 to 40 mg/kgon day 3, after which body temperature of the ZAG+Prop animals trackedthat of untreated. animals.

FIG. 38 is a pictorial diagram of a Western blot showing ZAG usinganti-mouse ZAG in mouse serum from mice treated with and without ZAG inthe absence or presence of propranonol. Endogenous murine ZAG increaseswith treatment by orally administered rhZAG, and such increase isblocked by propranolol.

FIG. 39 is a pictorial diagram of a Western blot showing ZAG usinganti-human ZAG against mouse serum from mice treated with and withoutZAG in the absence or presence of propranonol. Human ZAG is not detectedin mouse serum with or without propranolol.

FIG. 40 is a graphical diagram showing glucose levels during a glucosetolerance test in ob/ob mice treated with and without ZAG (p.o.) in theabsence or presence of propranolol.

FIG. 41 is a pictorial diagram of a Western blot showing ZAG in ob/obmice following oral administration. Treatment with rhZAG administeredorally causes an increase in endogenously expressed murine ZAG inplasma.

FIG. 42 is a pictorial diagram of a Western blot showing ZAG expressionin WAT from ob/ob mice treated with and without human ZAG (p.o.).

FIG. 43 is a pictorial diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice.

FIG. 44 is a pictorial diagram showing the effect of ZAG on signalingpathways in skeletal muscle of ob/ob mice.

FIG. 45 is a pictorial diagram showing the effect of ZAG on proteinsynthesis and degradation in skeletal muscle of ob/ob mice.

FIG. 46 is a pictorial diagram of a 14C ZAG autoradiograph showingstomach and plasma levels of ZAG from ob/ob mouse treated p.o., thesamples being taken 24-hours post treatment.

FIG. 47 is a graphical diagram showing weight change in ob/ob micetreated with and without 50 ug ZAG (p.o./gavage).

FIG. 48 is a graphical diagram showing glucose urine levels in ob/obmice treated with and without 50 ug ZAG (p.o./gavage).

FIG. 49 is a pictorial diagram of a Western blot. Anti-ZAG DiminishesAffects Caused by BRL37344 in vivo: Western blot of UCP3 in BAT of ob/obmice treated with and without BRL in the absence or presence ofAnti-ZAG. Treatment of ob/ob mice with BRL37344 causes an increase ofUCP3 in BAT, an effect which is blocked by the administration ofanti-ZAG antibodies.

FIG. 50 is a pictorial diagram of a Western blot. ZAG administeredorally to ob/ob mice causes an up-regulation of endogenous murine ZAG inplasma and in WAT. Western blot of mouse ZAG in p.o. rhZAG-dosed samplesof plasma(top) and WAT (bottom).

FIG. 51 is a pictorial diagram of a Western blot. Propranolol blocks theincrease in murine serum ZAG due to treatment with rhZAG p.o., but theadministered human ZAG is not found in plasma. Western blot of ZAG usingAnti-mouse ZAG in mouse serum from Mice treated with and without ZAG inthe absence or presence of propranonol (top). Human ZAG is not detectedin mouse serum. Western blot of ZAG using Anti-human ZAG in mouse serumfrom Mice treated with and without ZAG in the absence or presence ofpropranonol (bottom).

FIG. 52A is a graphical diagram showing the effect of ZAG concentrationon cyclic AMP production in CHO cells transfected with β3-AR (▪), β2-AR(□) and β3-AR (hashed). FIG. 52B is a graphical diagram showing effectof isoprenaline (10 μM) on cyclic AMP production in β1- (▪), β2- (□) andβ3-AR (hashed) transfected CHO cells in the absence or presence ofSR59230A (10 μM). Differences from basal levels of cyclic AMP areindicated as either a, p<0.05 or c, p<0.001.

FIG. 52C is a graphical diagram showing mRNA levels. Expression of β1-,β2- and β3-AR in CHO-K1 cells transfected with the respective humangenes as absolute numbers of β-AR mRNA molecules/m of total RNA,measured by RT—real time PCR (closed boxes) in comparison withexpression of GAPDH in the same sample (open boxes). FIG. 52D is agraphical diagram showing cyclic AMP production in CHO-K1 cellstransfected with human β1-, β2- and β3-AR in response to forskolin (20μM). (closed boxes) in relation to basal levels (open boxes).

FIGS. 52E, 52F and 52G is a graphical diagram showing specific bindingof ZAG to CHO-K1 cells transfected with human β1- (FIG. 52E), β2-(FIG.52F) and β3-AR (FIG. 52G) in the absence (▪) or presence (▴) of 100 uMnon-labelled ZAG Binding of similar concentrations of ZAG frozen andthawed(X) is also indicated.

FIG. 52H is a graphical diagram showing lipolytic activity of ZAG (0.58μM), either fresh (open), or frozen and thawed once (dashed), incomparison with isoprenaline (Iso; 10 μM)(solid) in murine epididymaladipocytes. Differences from control are shown as c, p<0.001, whiledifferences between fresh and frozen ZAG and in the presence of SR59230Aare shown as f, p<0.001.

FIG. 53A-53C are a graphical diagrams showing the effect of propanololon body weight (53A), body temperature (53B) and urinary glucoseexcretion (53C) in ob/ob mice treated with ZAG. Animals were dividedinto 4 groups (n=5 per group) to receive daily administration of ZAG (50mg, iv) (▪), ZAG+propanolol (40 mgkg⁻¹, po) (▴), while controls receivedeither PBS (♦) or PBS and propanolol (X). Differences from PBS are shownas c, p<0.001, while differences from ZAG alone are shown as f, p<0.001.

FIG. 53D is a pictorial diagram of liver histology after 60 dayscomparing control-treated and ZAG-treated example sections.

FIG. 54A are a graphical diagrams showing total areas under the glucosecurves (AUC) in arbitrary units and plasma glucose levels during aglucose tolerance test 3 days after initiation of ZAG (▪) in comparisonwith PBS (□) FIG. 54B is a graphical diagram showing plasma insulinlevels during the glucose tolerance test described in (FIG. 54A).

FIG. 54C is a graphical diagram showing glucose uptake into isolatedgastrocnemius muscle of ob/ob mice in the absence or presence or insulin(100 nM). Ob/ob mice were treated with ZAG with or without propanololfor 7 days prior to excision of muscle.

FIG. 54D is a graphical diagram showing glucose uptake into epididymaladipocytes of ob/ob mice in the absence or presence of insulin (10 nM).Animals received the treatments indicated for 7 days prior to excisionof WAT.

FIGS. 54E and 54F are graphical diagrams showing levels of TG (FIG. 54E)and NEFA (FIG. 54F) in ob/ob mice treated with PBS or ZAG, with orwithout propranolol for 7 days.

FIGS. 54G and 54H are pictorial diagrams of Western blots showing Glut 4expression in Gastronemius (54G) and WAT (54H) from ob/ob mice in thepresence of ZAG or Insulin or both. Differences from controls are shownas b, p<0.01 or c, p<0.001, while differences from ZAG alone are shownas d, p<0.05 or f, p<0.001. FIG. 55A is a pictorial diagram of a Westernblot.

FIGS. 55A, 55B and 55C are pictorial diagrams showing expression ofβ3-AR after treatment of ob/ob mice with ZAG (35 μg; i.v. day⁻¹) for 5days. Western blots showing expression of β3-AR in gastrocnemius muscle(54A), BAT (54B) and WAT (54C) of ob/ob mice treated with either PBS orZAG. The densitometric analysis is the average of three separate Westernblots. Differences from control as shown as c, p<0.001.

FIGS. 56A, 56B and 56C are pictorial diagrams of expression of β1- andβ3-AR in gastrocnemius muscle (56A), WAT (56B) and heart (56C) aftertreatment of ob/ob mice with ZAG (35 μg; i.v., daily) for 5 days.Differences from PBS treated animals is shown as a, p<0.05. FIGS. 57A,57B, 57C and 57D are pictorial diagrams of the effect of ZAG onexpression of uncoupling proteins. Western blots showing expression ofUCP1 showing expression of UCP1 in BAT (57A) and WAT (57B), andexpression of UCP3 in WAT (57C) and AMPK in gastrocnemius muscle (57D)in ob/ob mice after treatment with either PBS or ZAG (35 μg; i.v.,daily) for 5 days. The densitometric analysis is the average of threeseparate blots. Differences from PBS treated animals are shown as c,p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the observation that anti-humanZinc-α₂-glycoprotein (ZAG) antibodies reduce weight loss in models ofcachexia. As such, the invention provides methods for preventing weightloss in cachexia situations in a subject. Also provided arecombinatorial treatments to bring about a reduction in weight loss in asubject with cachexia.

Provided herein are formulations and methods for treating mammals and/orsupplementing a human or animal diet. The methods can include ingestingone or more of the described formulations for certain time periodsand/or in a certain order. Kits comprising one or more of theformulations are also provided. As such, the present invention is basedon the observation that recombinant zinc-α2-glycoprotein (ZAG) producesa decrease in body weight and increase in insulin responsiveness insubjects with no effect on food intake.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

The complete amino acid sequence of ZAG has been reported in a paperentitled “Complete amino acid sequence of human plasmaZinc-α₂-glycoprotein and its homology to histocompatibility antigens” byT. Araki et al. (1988) Proc. Natl. Acad. Sci. USA., 85, 679-683, whereinthe glycoprotein was shown as consisting of a single polypeptide chainof 276 amino acid residues having three distinct domain structures (A, Band C) and including two disulfide bonds together with N-linked glycansat three glycosylation sites. This amino acid sequence of thepolypeptide component is set out in FIG. 10 of the accompanyingdrawings. Although some subsequent publications have indicated that thecomposition of human ZAG can vary somewhat when isolated from differentbody fluids or tissues, all preparations of this material havesubstantially the same immunological characteristics. As reported by H.Ueyama, et al. (1991) “Cloning and nucleotide sequence of a humanZinc-α₂-glycoprotein cDNA and chromosomal assignment of its gene”,Biochem. Biophys. Res. Commun. 177, 696-703, cDNA of ZAG has beenisolated from human liver and prostate gland libraries, and also thegene has been isolated, as reported by Ueyama et al., (1993) “Molecularcloning and chromosomal assignment of the gene for humanZinc-α₂-glycoprotein”, Biochemistry 32, 12968-12976. H. Ueyama et al.have also described, in J. Biochem. (1994) 116, 677-681, studies on ZAGcDNAs from rat and mouse liver which, together with the glycoproteinexpressed by the corresponding mRNAs, have been sequenced and comparedwith the human material. Although detail differences were found as wouldbe expected from different species, a high degree of amino acid sequencehomology was found with over 50% identity with the human counterpart(over 70% identity within domain B of the glycoprotein). Again, commonimmunological properties between the human, rat and mouse ZAG have beenobserved.

The purified ZAG discussed above was prepared from fresh human plasmasubstantially according to the method described by Ohkubo et al. (Ohkuboet al. (1988) “Purification and characterisation of human plasmaZn-α₂-glycoprotein” Prep. Biochem., 18, 413-430). It will be appreciatedthat in some cases fragments of the isolated lipid mobilizing factor, ofZAG, or of anti-ZAG antibodies may be produced without loss of activity,and various additions, deletions or substitutions may be made which alsowill not substantially affect this activity. As such, the methods of theinvention also include use of functional fragments of anti-ZAGantibodies. The antibody or fragment thereof used in these therapeuticapplications may further be produced by recombinant DNA techniques suchas are well known in the art based possibly on the known cDNA sequencefor Zn-α₂-glycoprotein which has been published for example in H. Ueyamaet al. (1994) “Structure and Expression of Rat and Mouse mRNAs forZn-α₂-glycoprotein” J. Biochem., 116, 677-681. In addition, the antibodyor fragment thereof used in these therapeutic applications may furtherinclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring.

As used herein, ZAG polypeptides or proteins include variants of wildtype proteins which retain their biological function. As such, one ormore of the residues of a ZAG protein can be altered to yield a variantor truncated protein, so long as the variant retains it nativebiological activity. Conservative amino acid substitutions include, forexample, aspartic-glutamic as acidic amino acids;lysine/arginine/histidine as basic amino acids; leucine/isoleucine,methionine/valine, alanine/valine as hydrophobic amino acids;serine/glycine/alanine/threonine as hydrophilic amino acids.Conservative amino acid substitution also include groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. For example, it is reasonable to expect that replacementof a leucine with an isoleucine or valine, an aspartate with aglutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the properties of the resulting variant polypeptide.

Amino acid substitutions falling within the scope of the invention, are,in general, accomplished by selecting substitutions that do not differsignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. However, the invention also envisions variants withnon-conservative substitutions.

The term “peptide”, “polypeptide” and protein” are used interchangeablyherein unless otherwise distinguished to refer to polymers of aminoacids of any length. These terms also include proteins that arepost-translationally modified through reactions that includeglycosylation, acetylation and phosphorylation.

As discussed above, the present invention includes use of a functionfragment of a ZAG polypeptide or protein. A functional fragment, ischaracterized, in part, by having or affecting an activity associatedwith weight loss, lowering blood glucose level, increasing bodytemperature, improving glucose tissue uptake, increasing expression ofBet3 receptors, increasing expression of ZAG, increasing expression ofGlut 4, and/or increasing expression of UCP 1 and UCP 3. Thus, the term“functional fragment,” when used herein refers to a polypeptide thatretains one or more biological functions of ZAG. Methods for identifyingsuch a functional fragment of a ZAG polypeptide, are generally known inthe art.

As used herein, the term “antibody” includes reference to animmunoglobulin molecule immunologically reactive with a particularantigen, and includes both polyclonal and monoclonal antibodies. Theterm also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies) and heteroconjugateantibodies (e.g., bispecific antibodies). The term “antibody” alsoincludes antigen binding forms of antibodies, including fragments withantigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. Seealso, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.). See also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York (1998). The term also refers to recombinantsingle chain Fv fragments (scFv). The term antibody also includesbivalent or bispecific molecules, diabodies, triabodies, andtetrabodies. Bivalent and bispecific molecules are described in, e.g.,Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.(1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al.(1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, andMcCartney, et al. (1995) Protein Eng. 8:301.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs have beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody, in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” or a “V_(H)” refer to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, or Fab. References to “V_(L)” or a “V_(L)” refer to thevariable region of an immunoglobulin light chain, including the lightchain of an Fv, scFv, dsFv or Fab.

An antibody having a constant region substantially identical to anaturally occurring class IgG antibody constant region refers to anantibody in which any constant region present is substantiallyidentical, i.e., at least about 85-90%, and preferably at least 95%identical, to the amino acid sequence of the naturally occurring classIgG antibody's constant region.

As used herein, the term “monoclonal antibody” is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. Monoclonal antibodies useful with thepresent invention may be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow and Lane,“Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press,New York (1988); Hammerling et al., in: “Monoclonal Antibodies andT-Cell Hybridomas,” Elsevier, N.Y. (1981), pp. 563-681 (both of whichare incorporated herein by reference in their entireties).

Thus, in some embodiments, the antibodies of the invention may bechimeric, primatized, humanized, or human antibodies.

A “chimeric antibody” is an immunoglobulin molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, Science229:1202-1207 (1985); Oi et al., BioTechniques 4:214-221 (1986); Gillieset al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397, which are incorporated herein byreference in their entireties.

The term “humanized antibody” or “humanized immunoglobulin” refers to animmunoglobulin comprising a human framework, at least one and preferablyall complementarity determining regions (CDRs) from a non-humanantibody, and in which any constant region present is substantiallyidentical to a human immunoglobulin constant region, i.e., at leastabout 85-90%, and preferably at least 95% identical. Hence, all parts ofa humanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of one or more native humanimmunoglobulin sequences. Accordingly, such humanized antibodies arechimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. Often, frameworkresidues in the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. See, e.g., U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each ofwhich is incorporated by reference in its entirety). Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28:489-498 (1991);Studnicka et al., Prot. Eng. 7:805-814 (1994); Roguska et al., Proc.Natl. Acad. Sci. 91:969-973 (1994), and chain shuffling (U.S. Pat. No.5,565,332), all of which are hereby incorporated by reference in theirentireties.

In certain embodiments, completely “human” antibodies may be desirablefor therapeutic treatment of human patients. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods described above using antibody libraries derived from humanimmunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111;and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654;WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety. Human antibodies can also beproduced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. For an overview of this technology for producinghuman antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93(1995). For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporatedby reference herein in their entireties. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex (Princeton, N.J.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., Biotechnology 12:899-903(1988).

The term “primatized antibody” refers to an antibody comprising monkeyvariable regions and human constant regions. Methods for producingprimatized antibodies are known in the art. See e.g., U.S. Pat. Nos.5,658,570; 5,681,722; and 5,693,780, which are incorporated herein byreference in their entireties.

As used herein, the terms “epitope” or “antigenic determinant” refer toa site on an antigen to which an antibody binds. Epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

Antibodies of “IgG class” refers to antibodies of IgG1, IgG2, IgG3, andIgG4. The numbering of the amino acid residues in the heavy and lightchains is that of the EU index (Kabat, et al., “Sequences of Proteins ofImmunological Interest”, 5th ed., National Institutes of Health,Bethesda, Md. (1991); the EU numbering scheme is used herein).

Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, e.g., by oneor more injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include a protein, such as the polypeptide shown inSEQ ID NO: 1, encoded by a nucleic acid or a functional fragmentthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler & Milstein, Nature 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The immunizing agent will typically include a polypeptide asshown in SEQ ID NO: 1 or a functional fragment thereof.

Human antibodies can be produced using various techniques known in theart, including phage display libraries (Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J.Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be madeby introducing of human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg& Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments, the antibody is a single chain Fv (scFv). The V_(H)and the V_(L) regions of a scFv antibody comprise a single chain whichis folded to create an antigen binding site similar to that found in twochain antibodies. Once folded, noncovalent interactions stabilize thesingle chain antibody. While the V_(H) and V_(L) regions of someantibody embodiments can be directly joined together, one of skill willappreciate that the regions may be separated by a peptide linkerconsisting of one or more amino acids. Peptide linkers and their use arewell-known in the art. See, e.g., Huston et al., Proc. Nat'l Acad. Sci.USA 8:5879 (1988); Bird et al., Science 242:4236 (1988); Glockshuber etal., Biochemistry 29:1362 (1990); U.S. Pat. No. 4,946,778, U.S. Pat. No.5,132,405 and Stemmer et al., Biotechniques 14:256-265 (1993). Generallythe peptide linker will have no specific biological activity other thanto join the regions or to preserve some minimum distance or otherspatial relationship between the V_(H) and V_(L). However, theconstituent amino acids of the peptide linker may be selected toinfluence some property of the molecule such as the folding, net charge,or hydrophobicity. Single chain Fv (scFv) antibodies optionally includea peptide linker of no more than 50 amino acids, generally no more than40 amino acids, preferably no more than 30 amino acids, and morepreferably no more than 20 amino acids in length. In some embodiments,the peptide linker is a concatamer of the sequence Gly-Gly-Gly-Gly-Ser(SEQ ID NO:7), preferably 2, 3, 4, 5, or 6 such sequences. However, itis to be appreciated that some amino acid substitutions within thelinker can be made. For example, a valine can be substituted for aglycine.

Methods of making scFv antibodies have been described. See, Huse et al.,supra; Ward et al. supra; and Vaughan et al., supra. In brief, mRNA fromB-cells from an immunized animal is isolated and cDNA is prepared. ThecDNA is amplified using primers specific for the variable regions ofheavy and light chains of immunoglobulins. The PCR products are purifiedand the nucleic acid sequences are joined. If a linker peptide isdesired, nucleic acid sequences that encode the peptide are insertedbetween the heavy and light chain nucleic acid sequences. The nucleicacid which encodes the scFv is inserted into a vector and expressed inthe appropriate host cell. The scFv that specifically bind to thedesired antigen are typically found by panning of a phage displaylibrary. Panning can be performed by any of several methods. Panning canconveniently be performed using cells expressing the desired antigen ontheir surface or using a solid surface coated with the desired antigen.Conveniently, the surface can be a magnetic bead. The unbound phage arewashed off the solid surface and the bound phage are eluted.

ZAG and/or fragments thereof has been previously shown to bring about aweight reduction or reduction in obesity in mammals, as disclosed inU.S. Pat. Nos. 6,890,899 and 7,550,429, and in U.S. Pub. No.2010/0173829, the entire contents of each of which is incorporatedherein by reference. In one embodiment, the present inventiondemonstrates that anti-ZAG antibodies and/or functional fragmentsthereof reduces weight loss in models of cachexia. It is thereforecontemplated that the methods of the instant invention provide adetectable effect on symptoms associated with cachexia and/or diseasesassociated with muscle wasting disease.

Accordingly, in one aspect, the invention provides a method ofameliorating the symptoms of cachexia in a subject. The method includesadministering to the subject in need of such treatment a therapeuticallyeffective dosage of an inhibitor of the biological activity of apolypeptide having the sequence as shown in SEQ ID NO: 1. In oneembodiment, the treatment regimen may be for months (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months), or years. In another embodiment,the polypeptide is administered for a period of up to 21 days or longer.In another embodiment, the amelioration of symptoms is detectable withindays (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 3, or 4weeks), or months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months) of initiating treatment. In another embodiment, the treatmentregimen is about 10 days wherein there is amelioration of symptomsassociated with cachexia following treatment. In another embodiment, thetreatment regimen is about 21 days wherein there is amelioration ofsymptoms associated with cachexia following treatment.

In addition, it has been observed that a lipid mobilizing agent havingsimilar characteristics of ZAG and/or fragments thereof has also beenused to bring about a weight reduction or reduction in obesity inmammals, as disclosed in U.S. Published App. No. 2006/0160723,incorporated by herein by reference in its entirety. Finally, it hasbeen shown that ZAG and/or functional fragments thereof increases theinsulin responsiveness of adipocytes and skeletal muscle, and producesan increase in muscle mass through an increase in protein synthesiscoupled with a decrease in protein degradation regardless of whether aweight reduction or reduction in obesity is observed during treatment(see U.S. Ser. No. 12/614,289, incorporated herein by reference).

Additionally, β3 agonists are reportedly effective insulin sensitizingagents in rodents and their potential to reduce blood glucose levels inhumans has been a subject of investigation. Activation of β3 agonistsadrenoceptors stimulates fat oxidation, thereby lowering intracellularconcentrations of metabolites including fatty acyl CoA anddiacylglycerol, which modulate insulin signaling. Furthermore, it iscontemplated herein that certain β3 receptor agonists may not have foundsuccess in clinical trials given that one category of β3 receptorsavailable to these agents is located in the digestive system andparticularly in the mouth, pharynx, esophagus and stomach, resulting inminimal, if any, exposure of the agonist to most of these receptors.This theory is supported by the observation that several of the β3agonist therapeutic agents were found to be efficacious but had limitedbioavailability in the plasma space.

A number of formulations are provided herein. A formulation can be inany form, e.g., liquid, solid, gel, emulsion, powder, tablet, capsule,or gel cap (e.g., soft or hard gel cap). A formulation typically willinclude one or more compositions that have been purified, isolated, orextracted (e.g., from plants) or synthesized, which are combined toprovide a benefit (e.g., a health benefit in addition to a nutritionalbenefit) when used to supplement food in a diet.

In certain embodiments, recommended amounts per day or per serving of aformulation or of ingredients provided in a formulation may be set forthherein. In certain cases, as will be recognized by one having ordinaryskill in the art, one could vary the form of the formulation, e.g., bysubstituting a powder for a capsule, a tablet for a capsule, a gel-capfor a tablet, a gel-cap for a capsule, a powder for a gel-cap, or anysuch combination, in order to provide such recommended amounts per dayor per serving of a formulation.

Any of the formulations can be prepared using well known methods bythose having ordinary skill in the art, e.g., by mixing the recitedingredients in the proper amounts. Ingredients for inclusion in aformulation are generally commercially available.

Accordingly, in one aspect, the invention provides a formulation thatincludes ZAG or a functional fragment thereof. For example, the ZAG maybe mammalian ZAG, such human ZAG as shown in SEQ ID NO: 1, or fragmentsthereof. However, it should be understood that the ZAG may be derivedfrom any source provided that the ZAG retains the activity of wild-typeZAG. In one embodiment, the further includes a pharmaceuticallyacceptable carrier, which constitutes one or more accessory ingredients.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

Cachexia is commonly associated with a number of disease states,including acute inflammatory processes associated with critical illnessand chronic inflammatory diseases, cancer, AIDS, sepsis, COPD, renalfailure, arthritis, congestive heart failure, muscular dystrophy,diabetes, sarcopenia of aging, severe trauma (e.g., orthopedicimmobilization of a limb), metabolic acidosis, denervation atrophy, andweightlessness.

The term “therapeutically effective amount” or “effective amount” meansthe amount of a compound or pharmaceutical composition that will elicitthe biological or medical response of a tissue, system, animal or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician.

In some embodiments, the formulations of the invention are intended tobe orally administered daily. However, other forms of administration areequally envisioned. As used herein, the terms “administration” or“administering” are defined to include an act of providing a compound orpharmaceutical composition of the invention to a subject in need oftreatment. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually orally or by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. The phrases “systemic administration,”“administered systemically,” “peripheral administration” and“administered peripherally” as used herein mean the administration of acompound, drug or other material other than directly into the centralnervous system, such that it enters the subject's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration. As such, in one embodiment, the anti-ZAGantibodies, fragments thereof, and/or formulations of the invention areadministered to a subject via inhalation, intranasally, buccally,sublingually, intravenously, intramuscularly, and/or orally. In anotherembodiment, the antibodies or compositions thereof are formulated inrapid-melting compositions, extended release compositions, and the like.

As used herein, the term “ameliorating” or “treating” means that theclinical signs and/or the symptoms associated with cachexia are lessenedas a result of the actions performed. The signs or symptoms to bemonitored will be characteristic of cachexia and will be well known tothe skilled clinician, as will the methods for monitoring the signs andconditions. In addition to adipose/muscle mass loss, exemplary symptomsassociated with cachexia include, but are not limited to, fever,headache, chronic pain, body malaise, fainting, seizure associated withthe fever, shock, palpitations, heart murmur, gangrene, epistaxis,hemoptysis, cough, difficulty in breathing, wheezing, hyperventilationand hypoventilation, mouth breathing, hiccup and chest pain, abdominalpain, nausea or vomiting, heartburn, halitosis, and flatulence, ascompared to a normal subject or a subject that does not have cachexia.As such, an amelioration of the symptoms associate with cachexiaincludes but is not limited to, decreasing or reducing weight loss inthe subject and reversing one or more of the above-listed symptoms.

As used herein, the terms “reduce” and “inhibit” are used togetherbecause it is recognized that, in some cases, a decrease can be reducedbelow the level of detection of a particular assay. As such, it may notalways be clear whether the expression level or activity is “reduced”below a level of detection of an assay, or is completely “inhibited.”Nevertheless, it will be clearly determinable, following a treatmentaccording to the present methods, that amount of weight loss in asubject is at least reduced from the level prior to treatment.

ZAG has been attributed a number of biological roles, but its role as anadipokine regulating lipid mobilization and utilization is mostimportant in regulating body composition. Previous studies suggestedthat the increase in protein synthesis was due to an increase in cyclicAMP through interaction with the β-adrenoreceptor, while the decrease inprotein degradation was due to reduced activity of theubiquitin-proteasome proteolytic pathway. Studies in db/db mice showthat insulin resistance causes muscle wasting through an increasedactivity of the ubiquitin-proteasome pathway. An increasedphosphorylation of both PKR and eIF2α will reduce protein synthesis byblocking translation initiation, while activation of PKR will increaseprotein degradation through activation of nuclear factor-κB (NF-κB),increasing expression of proteasome subunits. In vitro studies usingmyotubes in the presence of high extracellular glucose showed thatactivation of PKR led to activation of p38MAPK and formation of reactiveoxygen species (ROS). p38MAPK can phosphorylate and activate cPLA₂ atSer-505 causing release of arachidonic acid, a source of ROS.Hyperactivation of p38MAPK in skeletal muscle has been observed inmodels of diet-induced obesity. In addition caspase-3 activity has beenshown to be increased in skeletal muscle of diabetic animals, which maybe part of the signaling cascade, since it can cleave PKR leading toactivation. Without being bound to theory, the ability of ZAG toattenuate these signaling pathways provides an explanation regarding itsability to increase muscle mass. As such, an anti-ZAG antibody isdemonstrated to decrease loss of muscle mass in cachexia situations.

Accordingly, in another aspect, the invention provides a method ofsupplementing a human or animal diet. The method includes administeringto the subject a ZAG polypeptide or a fragment thereof. In anotherembodiment, the method includes ingesting a formulation that includes aZAG polypeptide, such as the human ZAG polypeptide as set forth in SEQID NO: 1. A formulation can be ingested alone or in combination with anyother known formulation, in any order and for varying relative lengthsof time. In certain embodiments, certain formulations are used prior toother formulations, while other formulations are ingested concurrently.

Thus, ZAG is identified as a lipid mobilizing factor capable of inducinglipolysis in white adipocytes of the mouse in a GTP-dependent process,similar to that induced by lipolytic hormones. The data presented hereinsupports these findings by showing that ZAG has a similar lipolyticeffect in rat adipocytes, and, moreover, produces a decrease in bodyweight and carcass fat in mature male rats, despite the fact that thesequence homology between rat and human ZAG is only 59.4%.

ZAG also counters some of the metabolic features of the diabetic stateincluding a reduction of plasma insulin levels and improved response inthe glucose tolerance test. Thus, in another aspect, the inventionprovides a method of decreasing plasma insulin levels in a subject. Themethod includes administering to the subject a therapeutically effectivedosage of a polypeptide having the sequence as shown in SEQ ID NO: 1 ora fragment thereof. In one embodiment, the decrease in plasma insulinoccurs within 3 days of initiating treatment. In another embodiment, thetreatment regimen is administered for 10 days or longer. In anotherembodiment, the treatment regimen is administered for 21 days or longer.

In addition, ZAG has been shown to increase glucose oxidation andincrease the tissue glucose metabolic rate in adult male mice. Thisincreased utilization of glucose would explain the fall in both bloodglucose and insulin levels in ob/ob mice administered ZAG. Triglycerideutilization was also increased in mice administered ZAG, which wouldexplain the fall in plasma non-esterified fatty acids (NEFA) andtriglycerides (TG) despite the increase in plasma glycerol, indicativeof increased lipolysis. The increased utilization of lipid would beanticipated from the increased expression of UCP1 and UCP3 in BAT andUCP3 in skeletal muscle, resulting in an increase in body temperature.Thus, ZAG is identified as a lipid mobilizing factor capable of inducinglipolysis in white adipocytes of the mouse in a GTP-dependent process,similar to that induced by lipolytic hormones. As such, in oneembodiment, amelioration of the symptoms associated with hyperglycemiaalso includes an increase in body temperature of about 0.5° C. to about1° C. during treatment. In one embodiment, the increase in bodytemperature occurs within 4 days of initiating treatment. In anotherembodiment, amelioration of the symptoms associated with hyperglycemiaalso includes an increase in pancreatic insulin as compared topancreatic insulin levels prior to treatment, since less insulin isneeded to control blood glucose as a result of the presence of ZAG.

ZAG has also been shown to counter some of the metabolic features of thediabetic state including a reduction of plasma insulin levels andimproved response in the glucose tolerance test. In addition ZAGincreases the responsiveness of epididymal adipocytes to the lipolyticeffect of a β3-adrenergic stimulant. ZAG also increases the expressionof HSL and ATGL in epididymal adipose tissue which have been found to bereduced in the obese insulin-resistant state. Factors regulating theexpression of HSL and ATGL are not known. However, the specific ERKinhibitor, PD98059 downregulated HSL expression in response to ZAG,suggesting a role for MAPK in this process. Mice lacking MAPKphosphatase-1 have increase activities of ERK and p38MAPK in WAT, andare resistant to diet-induced obesity due to enhanced energyexpenditure. Previous studies have suggested a role for MAPK in theZAG-induced expression of UCP3 in skeletal muscle. ERK activation mayregulate lipolysis in adipocytes by phosphorylation of serine residuesof HSL, such as Ser-600, one of the sites phosphorylated by proteinkinase A.

The results presented herein show that ZAG administration to the ratalso increases the expression of ATGL and HSL in the rat. ATGL may beimportant in excess fat storage in obesity, since ATGL knockout micehave large fat deposits and reduced NEFA release from WAT in response toisoproterenol, although they did display normal insulin sensitivity. Incontrast HSL null mice, when fed a normal diet, had body weights similarto wild-type animals. However, expression of both ATGL and HSL arereduced in human WAT in the obese insulin-resistant state compared withthe insulin sensitive state, and weight reduction also decreased mRNAand protein levels.

Stimulation of lipolysis alone would not deplete body fat stores, sincewithout an energy sink the liberated NEFA would be resynthesized backinto triglycerides in adipocytes. To reduce body fat, ZAG not onlyincreases lipolysis, as shown by an increase in plasma glycerol, butalso increases lipid utilization, as shown by the decrease in plasmalevels of triglycerides and NEFA. This energy is channeled into heat, asevidenced by the 0.4° C. rise in body temperature in rats treated withZAG. The increased energy utilization most likely arises from theincreased expression of UCP1, which has been shown in both BAT and WATafter administration of ZAG. An increased expression of UCP1 would beexpected to decrease plasma levels of NEFA, since they are the primarysubstrates for thermogenesis in BAT. BAT also has a high capacity forglucose utilization, which could partially explain the decrease in bloodglucose. In addition there was increased expression of GLUT4 in skeletalmuscle and WAT, which helps mediate the increase in glucose uptake inthe presence of insulin. In mice treated with ZAG there was an increasedglucose utilization/oxidation by brain, heart, BAT and gastrocnemiusmuscle, and increased production of ¹⁴CO₂ from D-[U-¹⁴C] glucose, aswell as [¹⁴C carboxy]triolein (FIG. 24). There was also a three-foldincrease in oxygen uptake by BAT of ob/ob mice after ZAG administration.

While ZAG increased expression of HSL in epididymal adipocytes there wasno increase in either subcutaneous or visceral adipocytes. A similarsituation was observed with expression of adipose triglyceride lipase(ATGL). Expression of HSL and ATGL correlated with expression of theactive (phospho) form of ERK. Expression of HSL and ATGL in epididymaladipocytes correlated with an increased lipolytic response to the β3agonist, BRL37344. This result suggests that ZAG may act synergisticallywith β3 agonists, and suggests that anti-ZAG antibodies may actsynergistically with β3 antagonists.

As used herein, the term “agonist” refers to an agent or analog that iscapable of inducing a full or partial pharmacological response. Forexample, an agonist may bind productively to a receptor and mimic thephysiological reaction thereto. As used herein, the term “antagonist”refers to an agent or analog that does not provoke a biological responseitself upon binding to a receptor, but blocks or dampensagonist-mediated responses. The methods and formulations of theinvention may include administering anti-ZAG antibodies, or a functionalfragment thereof, in combination with a β3 antagonist, such as but notlimited to BRL37344, or a β3 agonist.

Examples of β3 agonists that may be used in the present inventioninclude, but are not limited to: epinephrine (adrenaline),norepinephrine (noradrenaline), isoprotenerol, isoprenaline,propranolol, alprenolol, arotinolol, bucindolol, carazolol, carteolol,clenbuterol, denopamine, fenoterol, nadolol, octopamine, oxyprenolol,pindolol, [(cyano)pindolol], salbuterol, salmeterol, teratolol,tecradine, trimetoquinolol, 3′-iodotrimetoquinolol,3′,5′-iodotrimetoquinolol, Amibegron, Solabegron, Nebivolol, AD-9677,AJ-9677, AZ-002, CGP-12177, CL-316243, CL-317413, BRL-37344, BRL-35135,BRL-26830, BRL-28410, BRL-33725, BRL-37344, BRL-35113, BMS-194449,BMS-196085, BMS-201620, BMS-210285, BMS-187257, BMS-187413, the CONH2substitution of SO3H of BMS-187413, the racemates of BMS-181413,CGP-20712A, CGP-12177, CP-114271, CP-331679, CP-331684, CP-209129,FR-165914, FR-149175, ICI-118551, ICI-201651, ICI-198157, ICI-D7114,LY-377604, LY-368842, KTO-7924, LY-362884, LY-750355, LY-749372,LY-79771, LY-104119, L-771047, L-755507, L-749372, L-750355, L-760087,L-766892, L-746646, L-757793, L-770644, L-760081, L-796568, L-748328,L-748337, Ro-16-8714, Ro-40-2148, (−)-RO-363, SB-215691, SB-220648,SB-226552, SB-229432, SB-251023, SB-236923, SB-246982, SR-58894A,SR-58611, SR-58878, SR-59062, SM-11044, SM-350300, ZD-7114, ZD-2079,ZD-9969, ZM-215001, and ZM-215967.

Examples of β-AR antagonists that may be used in the present inventioninclude, but are not limited to: propranolol, (−)-propranolol,(+)-propranolol, practolol, (−)-practolol, (+)-practolol, CGP-20712A,ICI-118551, (−)-buprranolol, acebutolol, atenolol, betaxolol,bisoprolol, esmolol, nebivolol, metoprolol, acebutolol, carteolol,penbutolol, pindolol, carvedilol, labetalol, levobunolol, metipranolol,nadolol, sotalol, and timolol.

Induction of lipolysis in rat adipocytes by ZAG is suggested to bemediated through a β3-AR, and the effect of ZAG on adipose tissue andlean body mass may also be due to its ability to stimulate the β3-AR.Induction of UCP1 expression by ZAG has been shown to be mediatedthrough interaction with a β3-AR. The increased expression of UCP 1 inWAT may also be a β3-AR effect through remodeling of brown adipocyteprecursors, as occurs with the β3-AR agonist CL316,243. Using knock-outmice the antiobesity effect of β3-AR stimulation has been mainlyattributed to UCP1 in BAT, and less to UCP2 and UCP3 through theUCP1-dependent degradation of NEFA released from WAT. Glucose uptakeinto peripheral tissues of animals is stimulated by cold-exposure, aneffect also mediated through the β3-AR. However, targeting the β3-AR hasbeen more difficult in humans than in rodents, since β3-AR play a lessprominent role than β1 and β2-AR subtypes in the control of lipolysisand nutritive blood flow in human subcutaneous abdominal adipose tissue.However, despite this the β3-AR agonist CL316,243 has been shown toincrease fat oxidation in healthy young male volunteers. This may be dueto the ability of β-adrenergic agonists to increase the number of β3-ARin plasma membranes from BAT.

Accordingly, in another aspect, the invention provides a method oftreating a subject to bring about a reduction in weight loss due tocachexia or a disease associated with muscle wasting. The methodincludes administering to the subject in need of such treatment atherapeutically effective dosage of a β3 antagonist in combination withan antibody, or a fragment thereof, that binds to the polypeptide havingthe sequence as shown in SEQ ID NO: 1. In another embodiment, the methodincludes administering to the subject in need of such treatment atherapeutically effective dosage of a β3-AR antagonist in combinationwith an antibody, or a fragment thereof, that binds to the polypeptidehaving the sequence as shown in SEQ ID NO: 1.

Recent results suggest that ZAG expression in adipose tissue may be moreimportant locally than circulating ZAG, by acting in a paracrine manner.Thus in humans, while mRNA levels of ZAG in visceral and subcutaneousfat correlated negatively with BMI, fat mass and insulin resistance,serum levels, determined by ELISA, correlated positively with parametersof adiposity (BMI and waist circumference) and insulin resistance. Thusthe ability of ZAG to induce its own expression in gastrocnemius muscle,WAT and BAT may be critical for its ability to increase lipolysis andenergy utilization.

In various embodiments of the invention, the purpose of combining ZAG,β-3AR agonists and β-AR antagonists varies depending on the purpose ofthe treatment and the status of the subject.

In one embodiment involving the treatment of obesity or diabetes inwhich it is desired to activate the β3AR mechanism to achieve thedesired lipolysis, glucose consumption, insulin sensitization, proteinsynthesis, increased energy expenditure, and the like. In thiscircumstance with some subjects it may be observed that the administeredZAG, or more likely the β-3AR agonist will exhibit some undesiredactivity at one or more of the β-1AR or the β-3AR, causing side effectsor diminishment of desired efficacy. This circumstance would then callfor the additional administration of β-AR antagonists, sometimesreferred to as “classic beta blockers” so as to prevent the undesiredactivity at the β-1AR or β-2AR. These β-AR antagonists would preferably,but not necessarily, be selected to block the receptor subtype (one ofβ-1AR, β-2AR) that is associated with the side effect or mitigation ofefficacy.

In another embodiment involving the treatment of cachexia. In cachexiacaused by different diseases, and within populations of subjects with agiven diseases, different degrees of cachexia are observed, and withdifferent proportions of muscle loss and fat loss.

In another aspect, a cachectic subject may be suffering from loss ofmuscle mass, but with either no loss of fat, or some degree of fat loss.Because muscle loss is typically a more clinically undesirable outcome,utilizing ZAG to cause some of the muscle build-up that occurs incachetic animals treated with ZAG, while also causing some degree of fatloss, may be desired. Thus treating such subjects with ZAG, a β-3ARagonist, and optionally as described above, β-AR antagonists, couldincrease muscle mass.

In another aspect, a cachectic subject may be suffering from loss of fatmass, with either no or some degree of loss of muscle mass. In thiscase, it may be desirable from a clinical standpoint to block the lossof fat, and so administration of antibodies specific to ZAG would beused, in order to block the action caused by ZAG and therefore decreasethe downstream action of β-3AR.

In another embodiment, involving treatment of lipidystrophy, in whichfat masses are disproportionate to the normal distribution within asubject, and in which loss of fat mass is desired. In this case, theadministration of one or more of ZAG, a β-3AR agonist and β-ARantagonist would be desired, with reasoning similar to the firstcircumstance.

All methods may further include the step of bringing the activeingredient(s) into association with a pharmaceutically acceptablecarrier, which constitutes one or more accessory ingredients. As such,the invention also provides pharmaceutical compositions for use intreating subjects having symptoms associated with cachexia. In oneembodiment, the composition includes as the active constituent atherapeutically effective amount of an anti-ZAG antibody as discussedabove, or a functional fragment thereof, together with apharmaceutically acceptable carrier, diluent of excipient.

Pharmaceutically acceptable carriers useful for formulating acomposition for administration to a subject are well known in the artand include, for example, aqueous solutions such as water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters.A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the conjugate. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. In addition, such physiologically acceptable compounds mayfurther be in salt form (i.e., balanced with a counter-ion such as Ca2+,Mg2+, Na+, NH4+, etc.), provided that the carrier is compatible with thedesired route of administration (e.g., intravenous, subcutaneous, oral,etc.). One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the physico-chemicalcharacteristics of the therapeutic agent and on the route ofadministration of the composition, which can be, for example, orally orparenterally such as intravenously, and by injection, intubation, orother such method known in the art. The pharmaceutical composition alsocan contain a second (or more) compound(s) such as a diagnostic reagent,additional nutritional substance, toxin, or therapeutic agent, forexample, a cancer chemotherapeutic agent and/or vitamin(s).

Formulations of the present invention may also include one or moreexcipients. Pharmaceutically acceptable excipients which may be includedin the formulation are buffers such as citrate buffer, phosphate buffer,acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols,ascorbic acid, phospholipids; proteins, such as serum albumin, collagen,and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomes;polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol; and glycine or other amino acids. Buffer systemsfor use with the formulations include citrate; acetate; bicarbonate; andphosphate buffers.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compoundin the form of a powder or granules; or as a suspension of the activecompound in an aqueous liquid or non-aqueous liquid such as a syrup, anelixir, an emulsion of a draught.

The nutritional supplement formulations can further include any numberof additional ingredients that are known to promote health and/or weightreduction. Exemplary additional ingredients include, but are not limitedto, low-glycemic ingredients such as carbohydrate sources, proteinsources and sources of dietary fiber. Such low-glycemic ingredients havebeen shown to curb appetite and cause a reduction in daily caloricintake.

An important macronutrient of the nutritional supplement is carbohydratebecause it has the greatest influence on satiety and subsequent weightloss. As used herein, satiety, refers to the sensation of fullnessbetween one meal and the next and satiation refers to a sensation offullness that develops during the progress of a meal and contributes tomeal termination. Foods with low-glycemic-indexes evoke a smaller risein blood glucose and insulin and a higher glucagon concentration, whichpromote satiety and prevent weight gain better than thosecarbohydrate-containing foods with higher ones because they take longerto digest and to be absorbed than carbohydrates withhigh-glycemic-indices.

The “glycemic index” is a system of predicting subsequent rises in bloodglucose after ingestion of carbohydrate-containing foods (Anderson, J.S. et al., Modern Nutrition in Health and Disease, ch. 70: 1259-86(1994); Wolever, T. M. S. et al., Am. J. Clin. Nutr., 54: 846-54 (1991);Wolever, T. M. S. et al., Diab. Care, 12: 126-32 (1990)). The glycemicindex characterizes the rate of carbohydrate absorption after a meal. Itis defined as the area under the glycemic response curve during a 2-hourperiod after consumption of 50 g of carbohydrate from a test fooddivided by the area under the curve of a standard, which is either whitebread or glucose. The glycemic index carbohydrates have the highest peakcirculating glucose in a 2 hour period following ingestion of food.Conversely, low-glycemic-index carbohydrates cause a lower peak glucoseand smaller area under the curve.

Many factors determine the glycemic index of foods. These includecarbohydrate type, fiber, protein and fat content and the method ofpreparation (overcooked foods evoke a higher response). Generallyhigh-glycemic-index carbohydrates are highly refined, and have arelatively high amount of glucose or starch compared to lactose, sucroseor fructose. Also, they are low in soluble fiber. The inclusion of fiberis important due to the way fiber facilitates weight loss by forming agel with the food in the stomach. This gelling action reduces the rateof gastric emptying and hence digestion rates which promote satiety.Other factors which affect satiety are the amount of carbohydrate, thecomplexity of the carbohydrate, and the other foods that are eatensimultaneously with the carbohydrate (e.g., fiber, protein, fat)(Ludwig, D. S., J Nutr., 130: 280S-3S (2000); Wolever, T. M. S. et al.,Am. J. Clin. Nutr., 54: 846-54 (1991); Wolever, T. M. S. et al., Diab.Care, 12: 126-32 (1990)). Bread and potatoes raise blood glucose morethan beans. Other foods containing no or non-digestible carbohydrateingested at the same time as carbohydrates (e.g., fat, fiber andprotein) reduces postprandial blood glucose and insulin levels (Wolever,T. M. S., et al., Am. J. Clin. Nutr., 54: 846-54 (1991)).

The low-glycemic-index carbohydrate source can be provided by a singlecarbohydrate or a combination. The carbohydrate source can furtherprovide a source of fiber and may be a natural sweetener, fructose,barley, konjac mannan, psyllium and combinations thereof. The proteinsource is of a high biological value and is selected from at least oneof the following: whey protein concentrate, casein, soy, milk, egg andcombinations of these. Additionally, the nutritional supplement maycontain, micronutrients, vitamins, minerals, dietary supplements (e.g.,herb), nutrients, emulsifiers, flavorings and edible compounds.

In one embodiment, the nutritional supplement formulation may furtherinclude a carbohydrate for sweetening the nutritional supplement.Exemplary carbohydrates useful for sweetening the nutritional supplementinclude, but are not limited to, fructose, evaporated cane juice,inulin, agave, honey, maple syrup, brown rice syrup, malt syrup, datesugar, fruit juice concentrate, and mixed fruit juice concentrate.

Dietary fiber that may be suitable for use in the invention includes butis not limited to cellulose, seeds, hemicellulose (e.g., bran, wholegrains), polyfructose (e.g., inulin and oligofructans), polysaccharidegums (e.g., Larch Arabinogalactan), oatmeal, barley, pectins, lignin,resistant starches. Examples of suitable fiber sources include but arenot limited to wheat bran, cellulose, oat bran, corn bran, guar, pectin,and psyllium.

Sources of protein can be any suitable protein utilized in nutritionalformulations and can include whey protein, whey protein concentrate,whey powder, egg, soy protein, soy protein isolate, caseinate (e.g.,sodium caseinate, sodium calcium caseinate, calcium caseinate, andpotassium caseinate), animal and vegetable protein and mixtures thereof.When choosing a protein source, the biological value of the proteinshould be considered first, with the highest biological values beingfound in caseinate, whey, lactalbumin, soy, delactosed milk solids, eggalbumin and whole egg proteins. These proteins have high biologicalvalue; that is, they have a high proportion of the essential aminoacids.

The nutritional supplement can also contain other ingredients such asone or a combination of other vitamins, minerals, antioxidants, fiber(e.g., ginkgo biloba, ginseng) and other nutritional supplements.Selection of one or several of these ingredients is a matter offormulation design, consumer and end-user preference. The amount ofthese ingredients added to the nutritional supplements of this inventionare readily known to the skilled artisan and guidance to such amountscan be provided by the RDA and DRI (Dietary Reference Intake) doses forchildren and adults. Vitamins and minerals that can be added include,but are not limited to, calcium phosphate or acetate, tribasic;potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodiumchloride); potassium chloride or acetate; ascorbic acid; ferricorthophosphate; niacin amide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolinate; potassium iodide; selenium; sodium selenate;sodium molybdate; phylloquinone; Vitamin D3; cyanocobalamin; sodiumselenite; copper sulfate; Vitamin A; Vitamin E; vitamin B6 andhydrochloride thereof; Vitamin C; inositol; Vitamin B12; potassiumiodide.

The amount of other ingredients per unit serving is a matter of designand will depend upon the total number of unit servings of thenutritional supplement daily administered to the patient. The totalamount of other ingredients will also depend, in part, upon thecondition of the patient. Preferably the amount of other ingredientswill be a fraction or multiplier of the RDA or DRI amounts. For example,the nutritional supplement will comprise 50% RDI (Reference DailyIntake) of vitamins and minerals per unit dosage and the patient willconsume two units per day.

Flavors, coloring agents, spices, nuts and the like can be incorporatedinto the product. Flavorings can be in the form of flavored extracts,volatile oils, chocolate flavorings (e.g., non-caffeinated cocoa orchocolate, or chocolate substitutes, such as carob), peanut butterflavoring, cookie crumbs, crisp rice, vanilla or any commerciallyavailable flavoring. Flavorings can be protected with mixed tocopherols.Examples of useful flavorings include but are not limited to pure aniseextract, imitation banana extract, imitation cherry extract, chocolateextract, pure lemon extract, pure orange extract, pure peppermintextract, imitation pineapple extract, imitation rum extract, imitationstrawberry extract, or pure vanilla extract; or volatile oils, such asbalm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, walnut oil,cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolateflavoring, vanilla cookie crumb, butterscotch or toffee. In a preferredembodiment, the nutritional supplement contains berry or other fruitflavors. The food compositions may further be coated, for example with ayogurt coating, if it is produced as a bar.

Emulsifiers may be added for stability of the final product. Examples ofsuitable emulsifiers include, but are not limited to, lecithin (e.g.,from egg or soy), and/or mono- and di-glycerides. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

Preservatives may also be added to the nutritional supplement to extendproduct shelf life. Preferably, preservatives such as potassium sorbate,sodium sorbate, potassium benzoate, sodium benzoate or calcium disodiumEDTA are used.

In addition to the carbohydrates described above, the nutritionalsupplement can contain artificial sweeteners, e.g., saccharides,cyclamates, aspartamine, aspartame, acesulfame K, and/or sorbitol. Suchartificial sweeteners can be desirable if the nutritional supplement isintended for an overweight or obese individual, or an individual withtype II diabetes who is prone to hyperglycemia.

The nutritional supplements of the present invention may be formulatedusing any pharmaceutically acceptable forms of the vitamins, mineralsand other nutrients discussed above, including their salts. They may beformulated into capsules, tablets, powders, suspensions, gels or liquidsoptionally comprising a physiologically acceptable carrier, such as butnot limited to water, milk, juice, sodas, starch, vegetable oils, saltsolutions, hydroxymethyl cellulose, carbohydrate. In one embodiment, thenutritional supplements may be formulated as powders, for example, formixing with consumable liquids, such as milk, juice, sodas, water orconsumable gels or syrups for mixing into other nutritional liquids orfoods. The powdered form has particular consumer appeal, is easy toadminister and incorporate into one's daily regimen, thus increasing thechances of patient compliance. The nutritional supplements of thisinvention may be formulated with other foods or liquids to providepremeasured supplemental foods, such as single serving breakfast bars,energy bars, breads, cookies, brownies, crackers, cereals, cakes, orbeverages, for example.

Thus, the nutritional supplement formulation may be administered as adietary supplement or as an additive to a consumable carrier such as afoodstuff. The composition may be incorporated into a foodstuff that islater cooked or baked. The components of the composition arestructurally stable to remain un-oxidized and are heat stable attemperatures required for baking or cooking.

To manufacture such a beverage, the ingredients are dried and madereadily soluble in water or other consumable liquids as described above.The beverage is a preferred nutritional supplement form due to itsability to aid in the sensation of satiety if consumed at least one halfhour prior to meals.

To manufacture such a food bar, the dry ingredients are added with theliquid ingredients in a mixer and mixed until the dough phase isreached; the dough is put into an extruder and extruded; the extrudeddough is cut into appropriate lengths; and the product is cooled.

For manufacture of other foods or beverages, the ingredients comprisingthe nutritional supplement of this invention can be added to traditionalformulations or they can be used to replace traditional ingredients.Those skilled in food formulating will be able to design appropriatefoods/beverages with the objective of this invention in mind.

The nutritional supplement can be made in a variety of forms, such aspuddings, confections, (i.e., candy), nutritional beverages, ice cream,frozen confections and novelties, or baked or non-baked, extruded foodproducts such as bars. In one embodiment, nutritional supplement is inthe form of a powder for a beverage or a non-baked extruded nutritionalbar.

In one embodiment, the consumable carrier is a meat product, such asnatural or cultured meat. In vitro meat, also known as cultured meat, isanimal flesh that has never been part of a complete, living animal. Theprocess of developing in vitro meat involves taking muscle cells andapplying a protein that helps the cells to grow into large portions ofmeat. Once the initial cells have been obtained, additional animalswould not be needed—akin to the production of yogurt cultures. In oneembodiment, the production of in vitro meat: loose muscle cells andstructured muscle, the latter one being vastly more challenging than theformer. Muscles consist of muscle fibers, long cells with multiplenuclei. Such cells do not proliferate by themselves, but arise whenprecursor cells fuse. Precursor cells can be embryonic stem cells orsatellite cells, specialized stem cells in muscle tissue. Theoretically,it is relatively simple to culture them in a bioreactor and then makethem fuse. For the growth of real muscle, however, the cells should grow“on the spot,” which requires a perfusion system akin to a blood supplyto deliver nutrients and oxygen close to the growing cells, as well asto remove the waste products. In addition, other cell types, such asadipocytes, need to be grown, and chemical messengers should provideclues to the growing tissue about the structure. Lastly, muscle tissueneeds to be physically stretched or “exercised” to properly develop(see, e.g., U.S. Pat. No. 6,835,390 and published Internationalapplication no. WO 99/31222, both of which are incorporated herein byreference). In yet another embodiment, the invention includes culturedmeat that is engineered to express ZAG in sufficient quantities suchthat addition of recombinant ZAG is unnecessary.

The ingredients can be administered in a single formulation or they canbe separately administered. For example, it may be desirable toadminister a bitter tasting ingredient in a form that masks its taste(e.g., capsule or pill form) rather than incorporating it into thenutritional composition itself (e.g., powder or bar). Thus, theinvention also provides a pharmaceutical pack or kit comprising one ormore containers filled with one or more of the ingredients of thenutritional compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by a governmentagency regulating the manufacture, use or sale of pharmaceutical ordietary supplement products, which notice reflects approval by theagency of manufacture, use of sale for human administration. The pack orkit can be labeled with information regarding mode of administration,sequence of administration (e.g., separately, sequentially orconcurrently), or the like. The pack or kit may also include means forreminding the patient to take the therapy. The pack or kit can be asingle unit dosage of the combination therapy or it can be a pluralityof unit dosages. In particular, the agents can be separated, mixedtogether in any combination, present in a formulation or tablet. Agentsassembled in a blister pack or other dispensing means is preferred.

In one embodiment, the formulation includes about 1.0 mg to 1000 mg ZAG.In another embodiment, the formulation includes about 1.0 mg to about500 mg ZAG. In another embodiment, the formulation includes about 1.0 mgto about 100 mg ZAG. In another embodiment, the formulation includesabout 1.0 mg to about 50 mg ZAG. In another embodiment, the formulationincludes about 1.0 mg to about 10 mg ZAG. In another embodiment, theformulation includes about 5.0 mg ZAG.

Accordingly, in another aspect, the invention provides the use ofanti-ZAG antibodies, or functional fragments thereof, as herein defined,for the manufacture of a medicament useful in human medicine fortreating symptoms and/or conditions associated with cachexia or diseasesassociated with muscle wasting disorders.

In one embodiment, the formulation of the present invention isadministered orally. In such embodiments, the formulation is at least70, 75, 80, 85, 90, 95 or 100% as effective as any other route ofadministration.

The total amount of formulation to be administered in practicing amethod of the invention can be administered to a subject as a singledose, either as a bolus or by ingestion over a relatively short periodof time, or can be administered using a fractionated treatment protocol,in which multiple doses are administered over a prolonged period of time(e.g., once daily, twice daily, etc.). One skilled in the art would knowthat the amount of formulation depends on many factors including the ageand general health of the subject as well as the route of administrationand the number of treatments to be administered. In view of thesefactors, the skilled artisan would adjust the particular dose asnecessary. In general, the formulation of the pharmaceutical compositionand the routes and frequency of administration are determined,initially, using Phase I and Phase II clinical trials.

Accordingly, in certain embodiments, the methods of the inventioninclude an intervalled treatment regimen. It was observed that long-termdaily administration of ZAG in ob/ob mice results in continuous weightloss. As such, in one embodiment, the treatment of ZAG or anti-ZAGantibodies, alone or in combination with one or more β-AR antagonists orβ3-AR agonists, is administered every other day. In another embodiment,the treatment is administered every two days. In another embodiment, thetreatment is administered every three days. In another embodiment, thetreatment is administered every four days.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1 Zinc-α₂-Glycoprotein Attenuates Hyperglycemia

To evaluate the ability of Zinc-α₂-glycoprotein (ZAG) to attenuateobesity and hyperglycemia ob/ob mice were administered ZAG which induceda loss of body weight, and arise in body temperature, suggesting anincreased energy expenditure. Expression of uncoupling proteins-1 and -3in brown adipose tissue were increased, while there was a decrease inserum levels of glucose, triglycerides and non-esterified fatty acids,despite an increase in glycerol, indicative of increased lipolysis.There was a decrease in plasma insulin and an improved response tointravenous glucose together with an increased glucose uptake intoadipocytes and skeletal muscle. Expression of hormone-sensitive lipasein epididymal adipocytes was increased. There was an increase inskeletal muscle mass due to an increase in protein synthesis anddecrease in degradation. This suggests that ZAG may be effective in thetreatment of hyperglycemia.

Dulbeccos' Modified Eagle's (DMEM) and Freestyle media were purchasedfrom Invitrogen (Paisley, UK) while fetal calf serum was from Biosera(Sussex, UK). 2-[1-¹⁴] Deoxy-D-glucose (sp.act.1.85GBq mmol⁻¹) andL-[2,6-³H] phenylalanine (sp.act.37Bq mmol⁻¹) were from AmericanRadiolabeled Chemicals (Cardiff UK). Rabbit polyclonal antibody tophospho (Thr-202) and total ERK1, total p38MAPK, phospho HSL (Ser-552),glucose transporter 4 (GLUT4), adipose triglyceride lipase, hormonesensitive lipase, and phospho PLA₂ (Ser-505) and to human ATGL werepurchased from Abcam (Cambridge, UK). Mouse monoclonal antibody to fulllength human ZAG was from Santa Cruz (Calif., USA), and mouse monoclonalantibody to myosin heavy chain type II was from Novacastra (via LeicaBiosystems, Newcastle, UK). Mouse monoclonal antibodies to 20Sproteasome α-subunits and p42 were from Affiniti Research Products(Exeter, UK). Mouse monoclonal antibody to phospho (Thr-180/Tyr-182)p38MAPK and rabbit polyclonal antisera to total and phospho (Thr-451)PKR, phospho (Ser-162) eIF2α and to total eIF2α were from New EnglandBiosciences (Herts, UK). Polyclonal rabbit antibodies to UCP1, UCP3 andtotal PKR and PHOSPHOSAFE™ Extraction Reagent were from Calbiochem (viaMerk Chemicals, Nottingham, UK). Peroxidase-conjugated goat anti-rabbitand rabbit anti-mouse antibodies were purchased from Dako (Cambridge,UK). Polyclonal rabbit antibody to mouse β-actin and the triglycerideassay kit were purchased from Sigma Aldrich (Dorset, UK). Hybond Anitrocellulose membranes and enhanced chemiluminescence (ECL)development kits were from Amersham Pharmacia Biotech (Bucks, UK). AWAKO colorimetric assay kit for NEFA was purchased from AlphaLaboratories (Hampshire, UK), and a mouse insulin ELISA kit waspurchased from DRG (Marburg, Germany). Glucose measurements were madeusing a Boots (Nottingham, UK) plasma glucose kit.

Production of Recombinant ZAG—

HEK293F cells were transfected with full length human ZAG cDNA in theexpression vector pcDNA 3.1, and maintained in FreeStyle medium under anatmosphere of 5% CO₂ in air at 37° C. ZAG was secreted into the medium,which was collected, and maximal protein levels (16 μgml⁻¹) wereobtained after 14 days of culture. To purify ZAG, media (200 ml) wascentrifuged at 700 g for 15 min to remove cells, and concentrated into avolume of 1 ml sterile PBS using an Amicon Ultra-15 centrifugal filterwith a 10 kDa cut-off. The concentrate (about 2 mg protein) was added to2 g DEAE cellulose suspended in 20 ml 10 mM Tris, pH 8.8 and stirred for2 h at 4° C. The DEAE cellulose bound ZAG and it was sedimented bycentrifugation (1500 g for 15 min) and the ZAG was eluted by stirringwith 20 ml 10 mM Tris, pH8.8 containing 0.3M NaCl for 30 min at 4° C.The eluate was washed and concentrated into a volume of 1 ml in sterilePBS using an Amicon centrifugal filter. The purified ZAG was free ofendotoxin, as determined with a LAL Pyrogent single test kit (Lonza,Bucks, UK).

Cell Culture and Purification of ZAG.

Single-cell suspensions of white adipocytes were prepared from mincedadipose deposits by incubation at 37° C. for 2 h in Krebs-Ringerbiocarbonate buffer containing 1.5 mgml⁻¹ collagenase, and 4% bovineserum albumin under an atmosphere of 95% oxygen: 5% CO₂ as previouslydescribed. For time-course studies adipocytes were suspended in DMEMcontaining 10% fetal calf serum at a concentration of 10⁵ cells ml⁻¹ andmaintained under an atmosphere of 10% CO₂ in air at 37° C. Human 293cells transfected with a plasmid containing human ZAG were seeded at aconcentration of 10⁵ cells ml⁻¹ in FreeStyle medium and maintained underan atmosphere of 5% CO₂ in air at 37° C. Maximal protein levels (16μgml⁻¹) were obtained after 14 days of culture. The media (200 ml) wasthen centrifuged at 700 g for 15 min to remove cells and concentratedinto a volume of 1 ml of sterile PBS using an Amicon Ultra-15centrifugal filter with a 10 kDa cut-off. After measurement of theprotein concentration of the sample (about 2 mg) it was added to 2 gDEAE cellulose suspended in 20 ml of 10 mM Tris, pH8.8 and stirred at 4°C. for 2 h. ZAG being negatively charged binds to the DEAE cellulose,which was sedimented by centrifugation (1500 g for 15 min), and elutedby stirring with 20 ml 10 mM Tris, pH8.8 containing 0.3M NaCl for 30 minat 4° C. The supernatant was washed and concentrated to a volume of 1 mlin sterile PBS using the Amicon centrifugal filter.

Animals—Mice.

Homozygous obese (ob/ob) mice from the colony maintained at AstonUniversity were used in the present study. The origin andcharacteristics of Aston ob/ob mice have been previously described. Malemice (20-21 weeks old, weight 90-100 g) were grouped into three per cagein an air conditioned room at 22±2° C. with a 12 h-light:12 h-dark cycleand fed a rat and mouse breeding diet (Special Diet Services, Witham,UK) and tap water ad libitum. They were administered ZAG (35 μg) in PBS(100 μl) b.d. by i.v. administration and body weight and food and waterintake were monitored daily. Control mice received PBS alone. Bodytemperature was measured daily by the use of a rectal thermometer (RSComponents, Northants, UK). All animal experiments were carried out inaccordance with the U.K. Animals (Scientific Procedures) Act 1986. Noadverse effects were observed after administration of ZAG.

Animal—Rats.

Mature male Wistar rats (one year old from our own colony) weighing540±82.5 g were housed individually and treated once daily i.v., witheither ZAG in PBS (100 μl) (50 μg per 100 g body weight), or with PBS(100 μl) as a control. Both food and water intake and body weight weremeasured daily. Animals were given free access to food (Special DietServices, Essex, UK) and water ad libitum. The animal experiment wascarried out under the welfare conditions imposed by the British HomeOffice. After 10 days treatment the animals were terminated and the bodycomposition determined. Animals were heated to 80-90° C. for 7 daysuntil constant weight was achieved. The water content was thendetermined from the difference between the wet and dry weight. Lipidswere extracted from the dry carcass using a sequence ofchloroform:methanol (1:1), ethanol/acetone (1:1) and diethyl ether (120ml of each) as described by Lundholm et al (14). The solvents wereevaporated and the fat weighed. The non-fat carcass mass was calculatedas the difference between the initial weight of the carcass and theweight of water and fat.

Lipolytic Assay.

Samples to be assayed were incubated with 10⁵ to 2×10⁵ adipocytes for 2h in 1 ml Krebs-Ringer bicarbonate buffer, pH 7.2. The concentration ofglycerol released was determined enzymatically by the method of Wieland(Wieland, 0. Glycerol UV method. In Methods of Enzymatic Analysis (ed.Bergmeyer, H. U.) (Academic Press, London, UK, pp 1404-1409, 1974)).Control samples containing adipocytes alone were analysed to determinethe spontaneous glycerol release. Activity was expressed as μmolglycerol released/10⁵ adipocytes/2 h.

Serum Metabolite Determinations.

Non-esterified fatty acids (NEFA) were determined using a Wako-ASC-ACODkit (Wako Chemical GmbH, Neuss, Germany). Triglycerides were determinedusing a Triglyceride kit (Sigma Chemical Co., Poole, United Kingdom) and3-hydroxybutyrate by a quantitative enzymatic determination kit (Sigma).Glucose was measured using a glucose analyser (Beckman, Irvine, Calif.)and glycerol was determined enzymatically using the method of Wieland asdescribed in “Methods of Enzymatic Analysis” (Ed. Bergmeyer, H. U.) Vol.3, pp 1404-1409, published by Academic Press, London (1974).

Isolation of Mouse Adipocyte Plasma Membranes.

In a typical procedure white adipocytes were isolated from mouseepididymal fat pads as referred to above except that the cells werewashed in 250 mM sucrose, 2 mM ethyleneglycolbis(β-aminoethylether)-N,N,N′, N′ (EGTA), 10 mM Tris-HCl (pH 7.4).Adipocytes were resuspended in 20 ml of the above buffer and homogenisedby aspirating through a Swinny filter at least 10 times. The cellhomogenate was then centrifuged at 300 g for 5 min, the fat cake removedfrom the surface and the remaining pellet and infranatant transferred toclean tubes. These were centrifuged at 30,000 g for 1 h at 4° C. and themembrane pellet formed was resuspended in the sucrose buffer (200 to 400μl). Plasma membranes were separated from other organelle membranes on aself-forming gradient of PERCOLL™ colloidial silica particles. Theconstituents were 250 mM sucrose, 2 mM EGTA, 10 mM Tris-HCl, pH 7.4;PERCOLL™; and 2M sucrose, 8 mM EGTA, 80 mM Tris-HCl, pH 7.4, mixed in aratio of 32:7:1 together with the membrane suspension (in a total volumeof 8 ml). This mixture was centrifuged at 10,000 g for 30 min at 4° C.The gradient was fractionated into 0.75 ml portions and each portion wasassayed for the presence of succinate dehydrogenase, NADH-cytochrome creductase, lactate dehydrogenase and 5′-nucleotidase to locate theplasma membrane fraction. The membrane fractions were resuspended in 150mM NaCl, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4 and centrifuged at 10,000 gat 4° C. for 2 min. The process was repeated twice. The washed plasmamembranes were then diluted in 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2mM EGTA and 4 μM phenylmethylsulfonyl fluoride (PMSF) at 1-2 mg/ml, snapfrozen in liquid nitrogen and stored at −70° C. until use.

Lipolytic Activity in Rat Adipocytes—

White adipocytes were prepared from finely minced epididymal adiposetissue of male Wistar rats (400 g) using collagenase digestion, asdescribed (Beck S A, et al. Production of lipolytic and proteolyticfactors by a murine tumor-producing cachexia in the host. Cancer Res47:5919-5923, 1987). Lipolytic activity was determined by incubating10⁵-2×10⁵ adipocytes for 2 h in 1 ml Krebs-Ringer bicarbonate buffer, pH7.2, and the extent of lipolysis was determined by measuring theglycerol released (Wieland 0. Glycerol UV method. Methods of EnzymaticAnalysis, edited by Bergmeyer H U. Academic Press, London, pp 1404-1409,1974). Spontaneous glycerol release was measured by incubatingadipocytes alone. Lipolytic activity was expressed as μmol glycerolreleased/10⁵ adipocytes/2 h.

Gel Electrophoresis. Gels were prepared according to the method ofLaemmli and generally consisted of a 5% stacking gel and a 15% SDS-PAGEresolving gel (denaturing or reducing conditions) or a 10% SDS-PAGEresolving gel (non-denaturing or non-reducing conditions). Samples wereloaded at 1-5 μg/lane. Bands were visualized by staining either withCoomassie brilliant blue R-250 or by silver. Samples were prepared forreducing conditions by heating for 5 min at 100° C. in 0.0625M Tris-HCl,pH 6.8, 10% glycerol, 1% SDS, 0.01% bromophenol blue and 5%2-mercaptoethanol.

Glucose Uptake into Adipocytyes.

Isolated adipocytes (5×10⁴) were washed twice in 1 ml Krebs-Ringerbicarbonate buffer, pH 7.2 (KRBS) and further incubated for 10 min atroom temperature in 0.5 ml KRBS containing 18.5MBq 2-[1-¹⁴C]deoxy-D-glucose and non-radioactive 2-deoxy-D-glucose to a finalconcentration of 0.1 mM. Uptake was terminated by the addition of 1 mlice-cold glucose-free KRBS, and the cells were washed three times with 1ml KRBS, lysed by addition of 0.5 ml 1M NaOH and left for at least 1 hat room temperature before the radioactivity was determined by liquidscintillation counting.

Glucose Uptake into Gastrocnemius Muscle—

Gastrocnemius muscles were incubated in Krebs-Henseleit bicarbonatebuffer for 45 min at 37° C. and then incubated for a further 10 min in 5ml Krebs-Henseleit buffer containing 185M Bq 2-[1-¹⁴C] deoxy-D-glucoseand non-radioactive 2-deoxy-D-glucose to a final concentration of 0.1mM. The muscles were then removed and washed in 0.9% NaCl for 5 minfollowed by dissolution in 0.5 ml 1M NaOH and the radioactivity wasdetermined by liquid scintillation counting.

Glucose Uptake into Soleus Muscle.

Soleus muscles were incubated in Krebs-Henseleit bicarbonate buffer for45 min at 37° C. and then incubated for a further 10 min in 5 mlKrebs-Henseleit buffer containing 185MBq 2-[1-¹⁴C] deoxy-D-glucose andnon-radioactive 2-deoxy-D-glucose to a final concentration of 0.1 mM.The muscles were then removed and washed in 0.9% NaCl for 5 min,followed by dissolution in 0.5 ml 1 MNaOH and the radioactivity wasdetermined by liquid scintillation counting.

Protein Synthesis and Degradation in Muscle.

The method for the determination of protein synthesis and degradation inmuscle has been previously described (Smith, K. L. & Tisdale, M. J.Increased protein degradation and decreased protein synthesis inskeletal muscle during cancer cachexia. Br. J. Cancer 67, 680-685(1993)). Gastrocnemius muscles were excised using ligatures andincubated for 30 min at 37° C. in RPMI 1640 medium lacking phenol redand saturated with O₂:CO₂ (19:1) and then washed with PBS. Proteinsynthesis was measured by the incorporation of L-[2,6-³H] phenylalanine(640 MBq) into acid-insoluble material using a 2 h period in which themuscles were incubated at 37° C. in RPMI/640 without phenol red andsaturated with O₂:CO₂ (19:1). Muscles were then rinsed innon-radioactive medium, blotted and homogenised in 2% perchloric acid.The rate of protein synthesis was calculated by dividing the amount ofprotein-bound radioactivity by the amount of acid soluble radioactivity.Protein degradation was determined by the release of tyrosine fromgastrocnemius muscle over a 2 h period in 3 ml of oxygenatedKrebs-Henseleit buffer, pH7.4, containing 5 mM glucose and 0.5 mMcycloheximide.

Measurement of Proteasome and Caspase Activity.

The ‘chymotrypsin-like’ activity of the proteasome was determinedfluorometrically by measuring the release of 7-amido-4-methylcoumarin(AMC) at an excitation wavelength of 360 nm and an emission wavelengthof 460 nm from the fluorogenic substrate N-succinyl Lys Lys Val Tyr.AMC(SEQ ID NO: 2) as previously described for myotubes (Whitehouse, A. S. &Tisdale, M. J. Increased expression of the ubiquitin-proteasome pathwayin murine myotubes by proteolysis-inducing factor (PIF) is associatedwith activation of the transcription factor NF-κB. Br. J. Cancer 89,1116-1122 (2003)). Gastrocnemius muscle was homogenised in 20 mM Tris,pH7.5, 2 mM ATP, 5 mM MgCl₂ and 50 mM DTT at 4° C., sonicated andcentrifuged at 18,000 g for 10 min at 4° C. to pellet insolublematerial, and the resulting supernatant was used to measure‘chymotrypsin-like’ enzyme activity in the presence or absence of theproteasome inhibitor lactacystin (10 ΞM). Only lactacystin suppressibleactivity was considered as true proteasome activity. The activity ofcaspase-3 was determined by the release of AMC fromAcAsp.Gly.Val.Asp.AMC (SEQ ID NO: 3), and the activity of caspase-8 wasdetermined by the release of 7-amino-4-trifluromethylcoumarin (AFC) fromthe specific substrate Z-Ile Glu Phe Thr Asp-AFC (SEQ ID NO: 4), usingthe supernatant from above (50 μg protein), and either the caspase-3 or-8 substrate (10 μM) for 1 h at 37° C., in the presence or absence ofthe caspase-3 (AcAspGluValAsp-CHO) (SEQ ID NO: 5) or caspase-8 (Ile GluPhe Thr Asp-CHO) (SEQ ID NO: 6) inhibitors (100 μM). The increase influorescence due to AFC was determined as above, while the increase influorescence due to AFC was measured with an excitation wavelength of400 nm and an emission wavelength of 505 nm. The difference in values inthe absence and presence of the caspase inhibitors was a measure ofactivity.

Western Blot Analysis.

Freshly excised gastrocnemius muscles were washed in PBS and lysed inPHOSPHOSAFE™ Extraction Reagent for 5 min at room temperature followedby sonication at 4° C. The lysate was cleared by centrifugation at18,000 g for 5 min at 4° C. and samples of cytosolic protein (5 μg) wereresolved on 12% sodium dodecyl suflate-polyacrylamide gelelectrophoresis at 180V for approximately 1 h. This was followed bytransference to 0.45 μm nitrocellulose membranes, which were thenblocked with 5% Marvel in Tris-buffered saline, pH 7.5, at 4° C.overnight. Both primary and secondary antibodies were used at a dilutionof 1:1000 except anti-myosin (1:250). Incubation was for 1 h at roomtemperature, and development was by ECL. Blots were scanned by adensitometer to quantify differences.

Samples of epididymal WAT, BAT and gastrocnemius muscle excised fromrats treated with ZAG or PBS for 5 days were homogenized in 0.25Msucrose, 1 mM HEPES, pH 7.0 and 0.2M EDTA, and then centrifuged for 10min at 4,500 rpm. Samples of cytosolic protein (10 μg) were resolved on12% sodium dodecylsulphate polyacrylamide gel electrophoresis and theproteins were then transferred onto 0.45 μm nitrocellulose membranes,which had been blocked with 5% Marvel in Tris-buffered saline, pH 7.5,at 4° C. overnight, and following four 15 min washes with 0.1% Tween inPBS, incubation with the secondary antibody was performed for 1 h atroom temperature. Development was by ECL.

Statistical Analysis.

The results are shown as means±SEM for at least three replicateexperiments. Difference in means between groups was determined byone-way analysis of variance (ANOVA) followed by the Tukey-Kramermultiple comparison test. P values less than 0.05 were consideredsignificant.

Results—Mice.

Purification of ZAG resulted in a product that was greater than 95% pure(FIG. 1A), confirmed as ZAG by immunoblotting (FIG. 1B). ZAG stimulatedlipolysis in epididymal adipocytes (FIG. 1D) but the lipolytic effectwas considerably reduced in adipocytes from both subcutaneous andvisceral deposits, although it was significantly elevated over basallevels (FIG. 1E). There was no significant difference in the extent ofstimulation of lipolysis between isoprenaline and ZAG in any adipocytegroup, although ZAG was more potent at inducing lipolysis thanisoprenaline on a molar basis. The effect of ZAG on the body weight ofob/ob mice over a 5 day period is shown in FIG. 1F. While controlanimals remained weight stable, animals treated with ZAG showed aprogressive weight loss, such that after 5 days there was a 3.5 g weightdifference between the groups, despite equal food (PBS 32±3.1 g; ZAG30±2.5 g) and water (PBS 140±8.2 ml; ZAG 135±3.2 ml) intake over thecourse of the experiment. There was a significant rise of bodytemperature of 0.4° C. after 4 days of ZAG administration (FIG. 1G),indicative of an increase in basal metabolic rate. Measurement of plasmametabolite levels suggest an increase in metabolic substrate utilizationin ZAG treated animals (Table 1). Thus there was a significant decreasein plasma glucose, triglycerides (TG) and non-esterified fatty acids(NEFA) in ZAG-treated animals, despite an increased glycerolconcentration indicative of an increased lipolysis. There was a 36%decrease in plasma insulin levels suggesting that ZAG is effective inreducing the diabetic state. ZAG mRNA levels in various tissues areshown in FIG. 1C.

TABLE 1 Plasma metabolite and insulin levels in ob/ob mice treated withZAG for 120 h PBS ZAG Glucose (mmol/L) 24.5 + 0.4  20.3 + 0.8 p < 0.01TG (mmol/L) 1.2 + 0.3  0.9 + 0.1 p < 0.05 Glycerol (μmol/L) 359 + 23  429 + 36 p < 0.001 Insulin (ng/mL) 41.2 + 0.6  26.3 + 0.52 p < 0.001BAT (g) 0.35 ± 0.09 0.73 ± 0.12 p < 0.01 NEFA (mEq/L)  0.6 + 0.12 0.23 +0.05 p < 0.001 Soleus (g) 0.52 ± 0.13 0.80 ± 0.09 p < 0.01 Gastrocnemius(g) 0.85 ± 0.12 1.12 ± 0.14 p < 0.01 Insulin Pancreas 4.52 ± 2.91 16.3 ±3.1 p = 0.0042 (pg/g pancerase)

To investigate this, a glucose tolerance test was performed, on fedanimals, after 3 days of ZAG administration (FIG. 2A). While bloodglucose levels were significantly elevated in PBS controls, there wasonly a small rise in ZAG treated animals, which remained significantlybelow the control group throughout the course of the study. In additionplasma insulin levels were significantly lower in ZAG treated animals atthe onset of the study and remained so during the 60 min of observation(FIG. 2B). ZAG administration increased glucose uptake into epididymal,visceral and subcutaneous adipocytes in the absence of insulin and alsoincreased glucose uptake into epididymal and visceral adipocytes in thepresence of low (1 nM) insulin (FIG. 2C). Glucose uptake intogastrocnemius muscle was also significantly enhanced in ZAG treatedanimals both in the absence and presence of insulin (100 nM) (FIG. 2D).The glucose uptake in gastrocnemius muscle of ZAG treated mice wasgreater than the response to insulin in non-treated animals.

ZAG administration also attenuated the effect of hyperglycemia onskeletal muscle atrophy. Thus ob/ob mice treated with ZAG showed asignificant increase in the wet weight of both gastrocnemius and soleusmuscles (Table 1). This was associated with over a two-fold increase inprotein synthesis in soles muscle (FIG. 3A), and a 60% decrease inprotein degradation (FIG. 3B). Gastrocnemius muscles from mice treatedwith ZAG showed a decreased activity of the proteasome‘chymotrypsin-like’ enzyme activity (FIG. 3C), which was notsignificantly different from that found in non-obese mice, and adecreased expression of both the 20S proteasome α-subunits (FIG. 3D),and p42, an ATPase subunit of the 19S regulator (FIG. 3E), suggesting areduced activity of the ubiquitin-proteasome pathway. Myosin levels wereincreased in ZAG-treated mice (FIG. 3F), while actin levels did notchange (FIG. 3G). In addition there was a reduction in the level ofphosphorylated forms of the dsRNA-dependent protein kinase (PKR) (FIG.4A) and eukaryotic initiation factor 2α (eIF2α) (FIG. 4B), which havebeen shown to be responsible for muscle atrophy induced by tumorcatabolic factors, and high levels of extracellular glucose. Otherenzymes in this pathway including phospholipase A₂ (PLA₂) (FIG. 4C), p38mitogen activated protein kinase (FIG. 4D) and caspases-3 and -8 (FIG.4E) were also attenuated in gastrocnemius muscles of ob/ob mice treatedwith ZAG. These changes were commensurate with a decrease in catabolicsignaling in muscle in response to ZAG.

ZAG, but not isoprenaline increased expression of phospho HSL inadipocytes which was completely attenuated by the extracellularsignal-regulated kinase (ERK) inhibitor PD98059¹⁴. While ZAG increasedexpression of HSL in epididymal adipocytes there was no increase ineither subcutaneous or visceral adipocytes (FIGS. 5B-5D). A similarsituation was observed with expression of adipose triglyceride lipase(ATGL) (FIGS. 5E-5G). Expression of HSL and ATGL correlated withexpression of the active (phospho) form of ERK (FIGS. 5H-5J). Expressionof HSL and ATGL in epididymal adipocytes correlated with an increasedlipolytic response to the β3 agonist, BRL37344 (FIG. 5K). This resultsuggests that ZAG may act synergistically with β3 agonists.

As previously reported, ZAG administration increased its expression inadipose tissue (FIG. 6A). ZAG expression remained elevated, for afurther 3 days in tissue culture in the absence of ZAG (FIG. 6B).Expression of HSL was also elevated in adipocytes for 3 days in tissueculture in the absence of ZAG (FIG. 6C). Administration of ZAG increasedthe expression of UCP1 (FIG. 6D) and UCP3 (FIG. 6E) in BAT (FIG. 6D) andUCP3 in skeletal muscle (FIG. 6F). An increased expression of uncouplingproteins would be expected to channel metabolic substrates into heat asobserved (FIG. 1G).

After 21 days, the plasma metabolite levels in the ob/ob mice wereobserved (Table 2), with monitored parameters shown in Table 3. Afurther drop in blood glucose (from 2.03 to 15.2 mM) and a rise inglycerol were observed, which seems greater since the control is lowerthan before. No change in NEFA, TG or insulin was observed at Day 21, ascompared to Day 5 (Table 1). It was noted that there is much moreinsulin in the pancreas in ZAG treated animals showing the drop inplasma insulin, which is not due to lower insulin production (e.g., aswould happen with a toxin to pancreatic beta cells), but rather due tothe fact that less insulin is needed to control blood glucose in the ZAGtreated animals.

TABLE 2 Plasma metabolite and insulin levels in ob/ob mice treated withZAG at Day 21. PBS ZAG Glucose (mmol/l) 24.1 ± 2.3 15.2 ± 2.1 p = 0.0085NEFA(mEq/l) 0.62 ± 0.008 0.22 ± 0.06 p = 0.0025 Glycerol  290 ± 25.2 450 ± 36.2 p = 0.0058 Triglycerides 1.72 ± 0.05 0.89 ± 0.08 p = 0.0072(mmol/l) Insulin (ng/ml) 39.5 ± 0.96 28.5 ± 0.34 p = 0.0056 InsulinPancreas  6.2 ± 3.2 14.5 ± 2.5 p = 0.0035 (pg/g pancerase)

TABLE 3 Parameters monitored in ob/ob mice treated with ZAG at Day 21.Parameter PBS ZAG p Start weight 92.5 ± 3.1  93.1 ± 1.9  Finish weight89.9 ± 1.3  83.95 ± 2.2  Food (g) 135 ± 6  145 ± 4  Water (ml) 268 ± 15 259 ± 20  BAT (g) 0.36 ± 0.21 0.41 ± 0.35 Gastrocnemius (g) 0.26 ± 0.150.39 ± 0.12 0.01 Soleus (g) 0.15 ± 0.06 0.18 ± 0.07

In addition, body temperature of the ob/ob mice increased 0.5° to 1° C.(FIG. 1G) within four days and peaked at 38.1° C. (FIG. 7) just beforethey lost the maximum amount of weight. This would correlate with theweight of brown adipose tissue which increases from 0.33±0.12 g in thecontrol to 0.52±0.08 g in the ZAG treated animals (FIG. 7). The weightof the gastrocnemius muscles was also increased from 0.2±0.05 g to0.7±0.1 g, while there was a progressive decrease in urinary glucoseexcretion (FIGS. 8A and 8B).

Results—Rats.

The lipolytic effect of human ZAG towards rat epididymal adipocytes incomparison with isoprenaline is shown in FIG. 11. At concentrationsbetween 233 and 700 nM ZAG produced a dose-related increase in glycerolrelease, which was attenuated by anti-ZAG monoclonal antibody, showingthe specificity of the action. The extent of lipolysis in rat adipocyteswas similar to that previously reported in the mouse. As in the mouse,the lipolytic effect of ZAG was completely attenuated by theβ3-adrenergic receptor (β3-AR) antagonist SR59230A, suggesting that theaction of ZAG was mediated through β3-AR. These results suggest that ZAGmay be effective in inducing fat loss in rats.

The effect of single daily i.v. injection of ZAG (50 μg/100 g b.w.) onthe body weight of mature male Wistar rats (540±83 g) is shown in FIG.12A. Compared with control rats administered the same volume of solvent(PBS), rats administered ZAG showed a progressive decrease in bodyweight, such that after 10 days, while rats treated with PBS showed a 13g increase in body weight, animals treated with ZAG showed a 5 gdecrease in body weight (Table 4). There was no difference in food (ZAG:102±32 g; PBS: 98±25 g) or water (ZAG: 135±35 ml; PBS: 125±25 ml) intakebetween the two groups during the course of the study, but ZAG-treatedanimals showed a consistent 0.4° C. elevation in body temperature, whichwas significant within 24 h of the first administration of ZAG (FIG.12B), indicating an elevated energy expenditure. Body compositionanalysis (Table 4) showed that the loss of body weight induced by ZAGwas due to a loss of carcass fat, which was partially offset by asignificant increase in lean body mass. There was a 50% increase inplasma glycerol concentration in rats treated with ZAG (Table 5),indicative of an increased lipolysis, but a 55% decrease in plasmalevels of non-esterified fatty acids (NEFA), suggesting an increasedutilisation. Plasma levels of glucose and triglycerides were alsoreduced by 36-37% (Table 5), also suggesting an increased utilization.There was a significant increase in the uptake of 2-deoxygluocse intoepididymal adipocytes of rats treated with ZAG for 10 days, which wasincreased in the presence of insulin (FIG. 12C). However, there was nosignificant difference in glucose uptake into adipocytes from ZAG or PBStreated animals in the presence of insulin (FIG. 12C). There was asmall, non-significant increase in glucose uptake into gastrocnemiusmuscle and BAT of rats treated with ZAG in comparison with PBS controls,but a significant increase in uptake in the presence of insulin (FIG.12D). These results suggest that the decrease in blood glucose is due toincreased, utilization by BAT, WAT and skeletal muscle, and this issupported by an increased expression of glucose transporter 4 (GLUT4) inall three tissues (FIG. 13).

TABLE 4 Body composition of male rats after treatment with either PBS orZAG Starting Final Weight weight weight change Water Fat Non fatTreatment (g) (g) (g) (g) (%) (g) (%) (g) (%) PBS 510 ± 30 523 ± 2 +13 ±3 326 ± 32 62 ± 2 105 ± 14  20 ± 3 90 ± 6  17 ± 3 ZAG 530 ± 45 525 ± 1 −5 ± 1 331 ± 5  63 ± 3 92 ± 5^(b) 18 ± 1 96 ± 2^(a) 18 ± 2 Differencesfrom animals treated with PBS are shown as a, p < 0.05 or b, p < 0.01

TABLE 5 Plasma metabolite and insulin levels in rats treated with eitherPBS or ZAG for 10 days Metabolite PBS ZAG Glucose (mmol/l) 25.5 ± 2.316.2 ± 2.1^(c) Trigylcerides (mmol/l) 1.75 ± 0.01  1.1 ± 0.09^(a)Glycerol (umol/l)  300 ± 52  450 ± 51^(c) NEFA (mEq/l) 0.58 ± 0.008 0.26± 0.06^(b) Differences from animals treated with PBS are shown as either^(a)p < 0.05; ^(b)p < 0.01 or ^(c)p < 0.001

ZAG administration increased expression of the uncoupling proteins(UCP)-1 and -3 in both BAT and WAT by almost two-fold (FIGS. 13A and13B), which would contribute to increased substrate utilization. In ratstreated with ZAG there was also an increased expression of the lipolyticenzymes adipose triglyceride lipase (ATGL) and hormone sensitive lipase(HSL) in epididymal adipose tissue (FIG. 15), again with a two-foldincrease. ATGL is mainly responsible for the hydrolysis of the firstester bond in a triacylglycerol molecule forming diacylgylcerol, whileits conversion to monacylglycerol is carried out by HSL. Expression ofZAG was also significantly increased in skeletal muscle, (FIG. 16A), WAT(FIG. 16B) and BAT (FIG. 16C) of rats treated with ZAG for 10 days,showing that exogenous ZAG boosts its own production in peripheraltissues.

There was a significant reduction in the expression of thephosphorylated forms of both dsRNA-dependent protein kinase (PKR) andeukaryotic initiation factor 2 (eIF2) on the α-subunit in gastrocnemiusmuscle of rats administered ZAG, while the total amount did not change(FIGS. 17A and 17B). Similar changes have been observed in ob/ob miceadministered ZAG (unpublished results) and were consistent with adepression of protein degradation and increase in protein synthesis inskeletal muscle.

Example 2 Interval Administration of Zinc-α₂-Glycoprotein

It was observed that long-term daily administration of ZAG in ob/ob miceresults in a cessation of weight loss. As such, it was determined that abreak of 3-4 days followed by re-infusion ZAG resulted in continuedweight loss and amelioration of the symptoms associated withhyperglycemia.

While not wanting to be limited by theory, it may be that the subjectsare receiving too much ZAG or that there is receptor desensitization asis seen with TNF. A pilot study was performed with 2 mice in each groupto determine optimal scheduling of ZAG delivery. An 8-10 g weight lossfrom a 90 g mouse was observed in about 3 weeks.

Adipocytes were removed from mice after 5 days of ZAG and theirresponsiveness to isoprenaline (iso) was measured after culture in theabsence of ZAG (FIG. 9). The responsiveness to iso is higher in ZAGtreated mice and this continues for a further 4 days (which was whenexpression of ZAG and HSL were increased) and then falls on day 5 (whenexpression was not increased) down to values of PBS control.

Example 3

Zinc-α₂-glycoprotein Attenuates Muscle Atrophy in ob/ob Mouse

This example demonstrates the mechanism by which ZAG attenuates muscleatrophy in the ob/ob mouse using a newly developed in vitro model(Russell et al, Exp. Cell Res. 315, 16-25, 2009). This utilizes murinemyotubes subjected to high concentrations of glucose (10 or 25 mM). Asshown in FIG. 18 high glucose stimulates an increase in proteindegradation (FIG. 18A), and depresses protein synthesis (FIG. 18B), andboth of these effects were completely attenuated by ZAG (25 μg/ml). Itwas therefore determined if the effect of ZAG was mediated through aβ3-AR using the antagonist SR59230A. However the SR compound (i.e.,SR59230A) can also act as a n-agonist, which it seemed to do in theseexperiments. Thus protein degradation induced by both 10 and 25 mMglucose was attenuated by both ZAG and the SR compound, and thecombination was additive rather than antagonistic (FIG. 19). For proteinsynthesis (FIG. 20) the SR compound seems to be similar to ZAG with noevidence of reversal, while with 10 mM glucose the SR compound causes anincrease in the depression of protein synthesis.

Example 4 Zinc-α₂-Glycoprotein Attenuates ROS Formation

It has been shown that formation of reactive oxygen species (ROS) isimportant in protein degradation induced by high glucose load. The datain FIG. 21 shows that ZAG completely attenuates the increase in ROSproduced by glucose, corresponding with the decrease in proteindegradation (FIG. 18A). High glucose also induces activation(phosphorylation) of PKR (FIG. 22A) and the subsequent phosphorylationof eIF2α(FIG. 22B) as is seen in skeletal muscle of ob/ob mice, whichwas also attenuated by ZAG. These results suggest that this in vitromodel will be useful to study how ZAG affects muscle mass at themolecular level.

Example 5 Zinc-α₂-Glycoprotein Increases Insulin Tolerance

An insulin tolerance test was also carried out in ob/ob miceadministered ZAG for 3 days (FIG. 23). Animals were administered twodoses of insulin (10 and 20 U/kg) by i.p. injection and blood glucosewas measured over the next 60 min. As can be seen (FIG. 23A) animalstreated with ZAG showed an increased sensitivity to insulin (10 U/kg)than those given PBS. At the higher concentration of insulin (20U/kg)this difference disappeared (FIG. 23B). The glucose disappearance curvefor 20 U/kg+PBS was almost identical to 10 U/kg+ZAG, so at this doselevel ZAG is reducing the requirement for insulin by 50%, but this canbe overcome by giving more insulin.

Example 6 Anti-Zinc-α₂-Glycoprotein Antibodies Reduce Weight Loss

The data shown in FIGS. 26-29 is from a study where the β3 agonist,BRL37344 was administered alone and in combination with an anti-ZAGantibody at 50 μg per day on a daily basis. Within 24 hours ofadministration, mice that were administered the antibody showedsignificant reduction in weight loss, as compared to mice administeredBRL37344.

Example 7 5-Day Administration of Zinc-α₂-Glycoprotein

A 5 day study was performed where ZAG was administered at 35 μg per dayi.v. on a daily basis for 5 days. At the end of the experiment tissueswere removed and blotted, or functional assays were carried out withisolated adipocytes. As can be seen in FIG. 6A, ZAG administrationincreased its expression in epididymal (ep), subcutaneous (sc) andvisceral (vis) fat about two-fold. When ep adipocytes were prepared andmaintained in tissue culture (RPMI 1640+10% FCS) ZAG expression wasmaintained for a further 3 days, even though no ZAG was added to theculture medium (FIG. 6B). In addition adipocytes from ZAG treated miceshowed an increased response to isoprenaline (10 μM), and this was alsomaintained for 4 days in tissue culture in the absence of ZAG (FIG. 9).The increased response to isoprenaline is due to an increased expressionof HSL by ZAG, and this was also maintained in tissue culture for 4 daysin the absence of ZAG (FIG. 6C). These results show that the effects ofZAG are maintained for a further 3 days when ZAG is withdrawn andtherefore it need not be administered on a daily basis. In fact, asdiscussed above, too much ZAG is more likely to lead to resistancerather than an increased response.

An increased expression of HSL was only seen in ep adipocytes after 5days ZAG (FIGS. 5B-5D), as was ATGL (FIGS. 5E-5G). There was an increasein expression of pERK only in ep adipose tissue (FIGS. 5H-5J), and aninhibitor of pERK (PD98059 10 μM) attenuated the increase in expressionof HSL in ep adipocytes incubated with ZAG for 3 h (FIG. 5A). ZAGincreased expression of UCP1 and UCP3 in BAT (FIGS. 6D and 6E) andmuscle (FIG. 6F) which would account for the increase in bodytemperature and fall in TG and NEFA in serum despite the increase inlipolysis.

Example 8 Role of β-Adrenergic Receptors in the Anti-Obesity andAnti-Diabetic Effects of Zinc-α₂-Glycoprotein

The goal of the study was to determine whether the β-adrenoreceptor(β-AR) plays a role in the anti-obesity and anti-diabetic effects ofzinc-α2-glycoprotein (ZAG). This has been investigated in CHO-K1 cellstransfected with the human β1-, β2-, β3-AR and in ob/ob mice. In CHO-K1cells transfected with the β3-AR the lowest concentration of ZAG tostimulate cyclic AMP production was 350 nM, while higher concentrations(580 nM) were required for cells transfected with the β2-AR, and therewas no increase in cyclic AMP in cells transfected with the β1-AR. Thiscorrelated with the Kd values for binding to the β3-AR (46±4 nM) andβ2-AR (71±2 nM), while there was no binding to the β1-AR. Freeze-thawingof ZAG, which destroyed its biological activity eliminated binding toβ2- and β3-AR. Treatment of ob/ob mice with ZAG increased proteinexpression of β3-AR in gastrocnemius muscle, and in white and brownadipose tissue, but had no effect on expression of β1- and β2-AR. Theeffect of ZAG on reduction of body weight and urinary glucose excretion,increase in body temperature, reduction in maximal plasma glucose andinsulin levels in the oral glucose tolerance test, and stimulation ofglucose transport into skeletal muscle and adipose tissue, wascompletely attenuated by the non-specific β-AR antagonist propanolol.These results evidence that the effect of ZAG on body weight and insulinsensitivity in ob/ob mice are manifested through a β-3AR, or possibly aβ2-AR.

Zinc-α₂-glycoprotein (ZAG) was first recognised to play a role in lipidmetabolism when tryptic fragments of a lipid mobilizing factor (LMF),thought to be responsible for loss of adipose tissue in cancer cachexia,were shown to be identical in amino acid sequence to ZAG. Both ZAG andLMF were shown to be immunologically identical, and both stimulatedlipolysis in murine adipocytes by the same amount, at the sameconcentration, by activation of adenylyl cyclase in a GTP-dependentprocess. Initial studies suggested that ZAG originated from the tumour,since tumours initiating cachexia showed high levels of expression,while other tumours which did not induce cachexia showed no expression.Later studies showed that ZAG was also produced in normal tissuesincluding liver, brown adipose tissue (BAT) and white adipose tissue(WAT), so that ZAG can be classified as an adipokine. Moreover, in bothcachectic mice and humans expression of ZAG mRNA in WAT was found to beincreased 10-fold and 2.7-fold respectively. In cachectic cancerpatients ZAG mRNA showed a negative correlation with body mass index(BMI), but a positive correlation with weight loss and serum glycerollevels. In contrast. ZAG mRNA levels in WAT have been shown to bedownregulated in obesity and correlated negatively with fat mass, BMI,plasma insulin and leptin. Treatment of ob/ob mice with ZAG decreasedbody weight and fat mass and improved the parameters of insulinresistance including decreasing plasma levels of glucose, insulin andnon-esterified fatty acids (NEFA), improving insulin sensitivity, andincreasing muscle mass. Serum ZAG levels have been found to besignificantly lower in mice fed a high fat diet than those fed a normaldiet, as well as in obese humans and mice. While ZAG overexpression inmice reduced both the body weight and weight of epididymal fat, ZAGknock-out animals showed an increased body weight, especially when fed ahigh fat diet. These results suggest that ZAG, like leptin, is closelyassociated with fat mass. However, while leptin is positively correlatedwith fat mass, ZAG is negatively correlated.

The lipolytic effect of ZAG was shown to be attenuated by theβ3-adrenoreceptor (β3-AR) antagonist, SR59230A, while LMF has been shownto bind to the β3-AR through a high affinity binding site (Kd 78±4.5nM). These results suggest that lipolysis mediated by ZAG is manifestedthrough a β3-AR. This study examined the role of the β-AR in the actionof ZAG, as well as determine the binding to β₁- and β₂-AR, and the roleof the β-AR in the anti-obesity and anti-diabetic effects of ZAG.

FCS (foetal calf serum) was from Biosera (Sussex, UK), while DMEM(Dulbecco's modified Eagle's medium) was from PAA (Somerset, UK) andFreestyle media and Superscript II reverse transcriptase were purchasedfrom Invitrogen (Paisley, UK). 2-[1-¹⁴C] deoxy-D-glucose sp.act. 1.85GBqmmol⁻¹) was purchased from American Radiolabeled Chemicals (Cardiff,UK). Na [¹²⁵I] (specific radioactivity >17 Ci mg⁻¹) was purchased fromPerkin Elmer Limited. Chicken polyclonal antibody to β3-AR and rabbitpolyclonal antibodies to β1-AR and β2-AR were purchased from Abcam(Cambridge, UK) and peroxidise-conjugated goat anti-chicken antibody wasfrom Santa Cruz (USA). Polyclonal rabbit antibodies to UCP1 and UCP3were from Calbiochem (via Merck Chemicals, Nottingham, UK).Peroxidase-conjugated goat anti-rabbit antibody was from Dako(Cambridge, UK). Polyclonal rabbit antibody to mouse β-actin,Tri-Reagent and propanolol were from Sigma Aldrich (Dorset, UK). HybondA nitrocellulose membranes were from GE Healthcare (Bucks, UK). TheParameter cyclic AMP assay kit was purchased from New England Biolabs(Hitchin, UK). The iodo beads and enhanced chemiluminescence (ECL)development kits were purchased from Thermo Scientific (Northumberland,UK). A mouse insulin ELISA kit was purchased from DRG (Marburg, Germany)and glucose measurements were made using a Boots (Nottingham, UK) plasmaglucose kit. Primers for reverse transcription and Easy-A one tubeRT.PCR system were from Agilent Technologies (Cheshire, UK).

Animals.

Obese (ob/ob) hyperglycaemic mice having an average weight of 71 g werebred. The background of these animals has been previously described(Bailey C J, et al. Influence of genetic background and age on theexpression of the obese hyperglycaemic syndrome in Aston ob/ob mice. IntJ Obes 6: 11-21, 1982), and they exhibit a more severe form of diabetesthan C57BL/6J ob/ob mice. Male mice (about 20 weeks of age) were groupedinto three per cage and kept in an air conditioned room at 22±2° C.,with ad libitum feeding of a rat and mouse breeding diet (Special DietServices, Witham, UK) and tap water. Mice were administered ZAG (50 μg,i.v. in 100 μl PBS) or PBS daily with or without propanolol (40 mgkg⁻¹,po, daily) and body weight and food and water intake were determined, aswell as urinary glucose excretion and body temperature, determined bythe use of a rectal thermometer (RS Components, Northants, UK). Aglucose tolerance test was performed on day 3. Glucose (1 gkg⁻¹ in avolume of 100 μl) was administered orally to animals which had beenfasted for 12 h. Blood samples were removed from the tail vein at 15,30, 60 and 120 min after glucose administration and used for themeasurements of glucose and insulin. At the end of the experiment theanimals were terminated by cervical dislocation, tissues removed andrapidly frozen in liquid nitrogen, and maintained at −80° C.

Production of Recombinant Human ZAG.

Human HEK293F cells, which had been transfected with pcDNA3.1 containinghuman ZAG were maintained in Freestyle medium, containing neomycin (50μgml⁻¹), under an atmosphere of 5% CO₂ in air. After 2 weeks of growthcells were removed by centrifugation (700 g for 15 min) and the mediumwas concentrated into a volume of 1 ml sterile PBS using an AmiconUltra-15 centrifugal filter with a M.W. cut-off of 10 kDa. The ZAG waspurified as described (Russell S T and Tisdale M J, Antidiabeticproperties of zinc-α2-glycoprotein in ob/ob mice. Endocrinol 151:948-957, 2010), by binding to DEAE cellulose, since ZAG has a highelectronegativity, and was eluted with 0.3 MNaCl. The ZAG produced bythis method was greater than 95% pure and was free of endotoxin, asdetermined by the LAL Pyrogent single test kit (Lonza). The purified ZAGwas stored at 4° C. in PBS.

[¹²⁵I] Labelling of ZAG.

One iodo bead that had been washed and dried was incubated with Na[¹²⁵I] (1 mCi per 100 μg protein) for 5 min in PBS, then ZAG (100 μgprotein) was added and left for a further 15 min. The reaction wasterminated by removal of the iodo bead, while free Na [¹²⁵I] was removedusing a Sephadex G25 column eluted with 0.1 MNaI. The [¹²⁵I] ZAG wasconcentrated against PBS using a Microcon microconcentrator with afilter cut-off of Mr 10,000. The specific activity of the [¹²⁵I] ZAG was8 Cimg protein¹.

Binding Studies and Cyclic AMP Determination.

CHO-K1 cells transfected with the human β1- and β2-AR obtained fromUniversity of Nottingham, UK, while CHOK1 cells transfected with theβ-AR were obtained from Astra Zeneca, Macclesfield, Cheshire, UK. Geneexpression was under the control of hygromycin, together with a β-galreporter construct, selected for resistance to G418. They weremaintained in DMEM supplemented with 2 mM glutamine, hygromycin B (50mgml⁻¹), G418 (200 mgml⁻¹), and 10% FCS, under an atmosphere of 10% CO₂in air. To determine the effect of agonists on cyclic AMP production,cells were grown in 24-well plates containing 1 ml of nutrient medium.ZAG or isoproterenol at the concentrations shown in FIG. 51 were addedto the cells and incubation was continued for 30 min. The medium wasremoved and replaced with 0.5 ml of 20 mM HEPES, pH 7.5, 5 mM EDTA and0.1 mM isobutylmethylxanthine, and the plates were heated on a boilingwater bath for 5 min and cooled on ice for 10 min. The concentration ofcyclic AMP was determined with an ELISA assay.

For binding studies cells were sonicated in 2 mM Tris HCl, pH7.5,containing 0.5M MgCl₂ and crude total membranes were pelleted bycentrifugation (13,000 g; 15 min) at 4° C. Binding studies were carriedout at 37° C. by incubating membranes (500 μg protein) in 0.4 ml 50 mMTris HCl, pH 7.5, containing 0.5 mM MgCl₂ for 60 min with variousconcentrations of [¹²⁵I] ZAG (3,000 to 15,000 cpm) in the absence orpresence of 100 μM non-labelled ZAG. The membranes were thenprecipitated by centrifugation at 13000 g for 20 min, the supernatantwas removed and the [¹²⁵I] bound to the pellet was quantitated using aPackard Corbra Model 5005 Auto-gamma counter. Binding was analysed usingnon-linear regression analysis (GraphPad Prism, Version 5.04). Specificbinding was regarded as the amount of labelled ZAG displaced bynon-radioactive ZAG.

RNA Isolation and RT-PCR._(—)

Quantitation of the mRNA transcripts for β1-, β2- and β3-AR in the threeCHO-K1 cells was based on the methodology already described (Moniotte S,et al. Real-time RT-PCR for the detection of beta-adrenoceptor messangerRNAs in small human endomyocardial biopsies. J Mol Cell Cardiol 33:2121-2133, 2001). Total RNA was extracted with Tri Reagent andquantitated by spectrophotometry, 800±34 ng total RNA was reversetranscribed, together with 2000 pmol random hexamers as primers usingSuperscript II reverse transcriptase at 43° C. for 50 min. The probesequences were selected to obtain T_(m)S approximately 10° C. lower thanthe matching primer pair. PCR was carried out using Easy-A one tubeRT.PCR system according to the manufacturer's instructions. The PCRconditions included a denaturing at 95° C. for 10 min, an annealing stepat 42-65° C., and an extension step at 68° C. for 2 min, and with afinal extension at 68° C. for 10 min. There were 40 cycles ofamplification. Expression of β-AR mRNA was determined by the Δ-CT methodusing Stratagenes MxPro, QPCR software v3.00.

Western blot analysis. WAT, BAT, heart and gastrocnemius muscle werethawed, washed in PBS, and lysed in Phosphsafe™ Extraction reagent for 5min at room temperature, followed by sonication at 4° C. The supernatantformed by centrifugation at 18,000 g for 5 min at 4° C. was used forWestern blotting. Cytosolic protein (5 μg for UCP's and 20 μg for β-AR)was resolved on 12% sodium dodecylsulphate polyacrylamide gels byelectrophoresis at 180V for about 1 h and transferred on to 0.45 μmnitrocellulose membranes, which had been blocked with 5% (w/v) non-fatdried milk (Marvel) in Tris-buffered saline, pH 7.5, at 4° C. overnight.Prior to adding the primary antibodies membranes were washed for 15 minin 0.1% Tween 20-buffered saline. Both primary and secondary antibodieswere used at a dilution of 1:1000. Incubation was for 1 h at roomtemperature and development was by ECL. Blots were scanned by adensitometer to quantify differences.

Glucose Uptake into Adipose Tissue and Skeletal Muscle.

Uptake of 2-[1-¹⁴C] deoxy-D-glucose (2-DG) into freshly isolatedepididymal adipocytes and gastrocnemius muscle was determined aspreviously described (Russell S T and Tisdale M J, Antidiabeticproperties of zinc-α2-glycoprotein in ob/ob mice. Endocrinol 151:948-957, 2010).

Statistical Analyses.

Results are shown as mean±SEM for at least three replicate experiments.Differences in means between groups was determined by one-way analysisof variance (ANOVA) followed by Tukey-Kramer multiple comparison test. pvalues <0.05 were considered significant.

The effect of human ZAG on cyclic AMP production in CHO cellstransfected with human β1, β2 and β3-AR is shown in FIG. 51. At lowconcentrations (up to 460 nM) there was specific stimulation of cyclicAMP production only in cells transfected with the (β3-AR (FIG. 51A).However, at 580 nM there was also a significant increase in cyclic AMPlevel in CHO cells transfected with the β2-AR, although the magnitude ofthe change was less than in cells transfected with the β3-AR. There wasno increase in cyclic AMP in CHO cells transfected with the β1-AR at anyconcentration of ZAG (FIG. 51A). In contrast isoprenaline (10 μM) showedsignificant increases in cyclic AMP level in CHO cells transfected withβ1-, β2- and β3-AR, showing the lack of specificity to the threeisoforms of the β-AR (FIG. 51B). The increase in cyclic AMP byisoprenaline through β1-, β2- and β3-AR was attenuated by SR59230A,showing a lack of specificity of this agent to the β3-AR.

To determine whether expression of the β3-AR was the same in the threecell lines mRNA levels of β1-AR, β2-AR and β3-AR was determines byRT-PCR as described (Moniotte S, et al. Real-time RT-PCR for thedetection of beta-adrenoceptor messanger RNAs in small humanendomyocardial biopsies. J Mol Cell Cardiol 33: 2121-2133, 2001). Thedata in FIG. 51C show that the level of expression of each β-AR is thesame in relation to the housekeeping gene GAPDH. Moreover, the level ofadenylate cyclase, as determined by cyclic AMP production in thepresence of forskolin, was also similar in the three cell lines (FIG.51D). These results suggest that a comparison between the β-AR in thethree cell lines is valid.

The affinity of binding of ZAG to the three β-AR was determined using¹²⁵I labelled ZAG and crude membranes from CHO-K1, β1, β2 and β3 cells(FIGS. 51E, F and G). The data was evaluated using non-linear regressionanalysis and the Kd and Bmax values are shown. The binding data reflectthe stimulation of cyclic AMP production by ZAG as shown in FIG. 51A.Thus ZAG bound predominantly to β3-AR (high Bmax and lowest Kd), less soto β2-AR (Bmax 20% of β3-AR and Kd twice β3-AR), and not at all to β1-AR(no Bmax and high Kd). Non-specific binding was determined by thebinding of [¹²⁵I] ZAG in the presence of 100 μM non-labelled ZAG, andthese values were subtracted from the total binding to give the specificbinding values. The lipolytic activity of ZAG was shown to be destroyedby a single freezing and thawing cycle (FIG. 51H), probably due to achange in conformation of the protein. To determine whether thisdisrupted binding to β-AR two experiments were performed: (i)Freeze-thaw [¹²⁵I] ZAG was used in the binding studies, which completelyattenuated binding to the β2- and β3-AR (FIGS. 51F and G). (ii)Freeze/thawed non-labelled ZAG was used in a competition assay with[¹²⁵I]ZAG, as in the determination of non-specific binding above. Incontrast with fresh non-labelled ZAG this had no effect on either the Kdor Bmax for binding to β2-AR or β3-AR. These results suggest thatfreeze/thawing ZAG destroys biological activity by preventing binding toβ-AR.

To determine whether the effects of ZAG on body weight and insulinsensitivity were due to interaction with a β-AR, ob/ob mice were treatedwith ZAG (50 μg, iv, daily), as previously reported (Russell S T andTisdale M J, Antidiabetic properties of zinc-α2-glycoprotein in ob/obmice. Endocrinol 151: 948-957, 2010), in the absence and presence of thenon-specific β-AR antagonist propanolol (40 mg kg⁻¹, po, daily). Thisdose level is higher than that commonly employed with β2-AR agonists,since higher levels are required to counteract the effect of β3-ARagonists (Liu Y L and Stock M J, Acute effects of the beta3-adrenoreceptor agonist, BRL 35135, on tissue glucose utilisation. Br JPharmacol. 114: 888-894, 1995). Propanolol completely attenuated thedecrease in body weight produced by ZAG (FIG. 52A), although animalstreated with propanolol alone did not show such a large weight gain asdid PBS controls. As previously reported (Russell S T and Tisdale M J,Antidiabetic properties of zinc-α2-glycoprotein in ob/ob mice.Endocrinol 151: 948-957, 2010) mice treated with ZAG showed an increasedbody temperature (FIG. 52B), and this was completely attenuated bypropanolol, as was the reduction in the urinary excretion of glucose(FIG. 52C). ZAG alone had no effect on liver lipids, although there wassome increase in glycogen (FIG. 52D). Propanolol also blocked thereduction in peak plasma glucose levels, and the area under the glucosecurve (AUC) induced by ZAG in the oral glucose tolerance test (FIG.53A), as well as the corresponding reduction in peak plasma insulinlevels (FIG. 53B). Animals treated with ZAG showed an increased glucoseuptake into gastrocnemius muscle in the presence of insulin (10 nM)(FIG. 53C), and this was completely attenuated in gastrocnemius musclefrom mice receiving propanolol. Epididymal adipocytes from mice treatedwith ZAG also showed an enhanced glucose uptake in the absence andpresence of insulin (FIG. 53D), and this was also completely attenuatedin animals treated with propanolol. The decrease in serum levels oftriglycerides (TG) and non-esterified fatty acids (NEFA) produced by ZAGwere also attenuated by propranolol (FIGS. 53E and F). These resultssuggest that the biological effects of ZAG are mediated through a β-AR.To determine whether ZAG can increase insulin signalling the effect onGlut4 expression was determined. Both insulin and ZAG increasedexpression of Glut4 in gastrocnemius muscle (FIG. 53G) and WAT (FIG.53H), but the combination did not produce an increase over that ofinsulin alone. These results suggest that ZAG influences the samesignalling pathways as insulin, but does not increase insulinsignalling.

A number of β3-agonists are known to increase expression of the β3-AR.To determine whether ZAG had the same effect, tissue β3-AR expressionwas quantified by Western blotting after 5 days of treatment of ob/obmice with ZAG. The results in FIG. 54 show a two-fold increase in β3-ARexpression in gastrocnemius muscle (FIG. 54A) an 89% increase in BAT(FIG. 54B) and a 85% increase in WAT (FIG. 54C). In contrast there wasno change in expression of β1-AR or β2-AR in either gastrocnemius muscle(FIG. 55A) or WAT (FIG. 55B) and no change in expression of β1-AR inheart (FIG. 55C), but a small increase in β2-AR which just reachedsignificance (FIG. 55C).

The increased expression of the β3-AR in BAT and WAT (FIG. 54) would beexpected to lead to an increased expression of UCP1, which is observedin both BAT (FIG. 56A) and WAT (FIG. 56B) after ZAG administration. Invitro experiments have shown that induction of expression of UCP3 by ZAGwas attenuated by the mitogen activated protein kinase (MAPKK) inhibitorPD98059, suggesting the involvement of MAPK in this process. Previousstudies have shown an increase in expression of ERK in WAT ofZAG-treated mice. This would be expected to lead to an increase inexpression of UCP3 in WAT, as was observed (FIG. 56C). It was alsopreviously reported that an increase in UCP3 in skeletal muscle of ob/obmice after administration of ZAG. The increased expression of UCP'swould provide a sink for the NEFA released from adipose tissue,generating heat as previously reported.

In addition treatment with ZAG produced an increase in expression ofAMPK in skeletal muscle (FIG. 56D), which would lead to an increaseoxidation of long-chain fatty acids, decreasing the availability for thesynthesis of triglycerides, as well as stimulating glucose uptakethrough increased expression of GLUT4.

This study has found that ZAG binds predominantly to the β3-AR, withintermediate binding to the β2-AR, and no binding to the β1-AR. Thehuman β3-AR is 51% homologous in amino acid sequence to the β₁-AR and46% homologous to the β3-AR. The Bmax for ZAG binding to the β3-AR isabout three times that for the β2-AR, while the Kd is about half. The Kdfor ZAG for binding to the β3-AR is about 100-fold lower than that ofCGP 12177, a partial agonist, while the Bmax is only slightly lower.These results were obtained using [¹²⁵I] ZAG which may havenon-equivalent binding activity to native ZAG, which could lead to overor under estimation of the Kd. While most of the studies with ZAG havebeen carried out in rodents there is a difference between human androdent β3-AR. Thus BRL37344 is less effective at stimulating adenylylcyclase via human than rodent β3-AR, while CGP12177 is an effectiveagonist at human β3-AR, but a poor partial agonist at the rat β3-AR. HowZAG binds to the β3-AR and β2-AR, while not binding to the β1-AR, is notknown, but the conformation of the protein is very important, sincebinding is destroyed by a single freeze/thaw cycle, as is also theability of ZAG to stimulate lipolysis in murine adipocytes. The Kdvalues are in the range expected, both from studies on the stimulationof lipolysis, and cyclic AMP production by ZAG. However, they are morethan 10-times lower than the human plasma concentration reported usingan ELISA (600 nM), but are comparable with that reported using massspectrometry (85 nM). If the former value was true ZAG would bemaximally stimulating the β3- and β3-AR at normal plasma concentrations,which is clearly not correct. Care must be taken in interpreting plasmaconcentrations of ZAG using an ELISA, since there may be othercomponents which bind to the anti-ZAG antibody, giving apparently higherconcentrations. Thus ZAG has been shown to non-specifically bind to amonoclonal antihuman erythropoietin antibody giving apparently highervalues in samples containing increased amounts of urinary ZAG.

The effect of ZAG on obesity and diabetes in the ob/ob mouse model maybe due to its ability to bind to β3-AR. Thus β3-AR agonists showanti-obesity effects in rodent models similar to ZAG, which induced anincreased mobilisation of triglycerides from WAT depots, increased fatoxidation, and increased BAT-mediated thermogenesis, resulting in aselective reduction in body fat and preservation of fat-free mass. Aswith ZAG the anti-diabetic effects of β3-AR agonists are independent ofthe anti-obesity effects, and occur at dose levels which do not induceweight loss. Treatment of ob/ob mice with the β3-AR agonist BRL 35135normalised plasma glucose levels and significantly decreased plasmainsulin and non esterified fatty acid (NEFA) levels. As with ZAG BRL35135 stimulated glucose uptake into three types of skeletal muscle,BAT, WAT, heart and diaphragm, which was independent of the action ofinsulin. Another β3-agonist L-796568 increased lipolysis and energyexpenditure in obese men when administered as a single dose. However,treatment for 28 days had no major lipolytic or thermogenic effect,although it lowered triacylglycerol concentration. This may be due toinsufficient recruitment of β3-AR responsive tissues in humans, ordown-regulation of β3-AR with chronic dosing. Studies in humansubcutaneous abdominal adipose tissue show that β3-AR play a weaker rolein the control of lipolysis than found in rodents, and that mobilisationof lipids is mainly through β1 and β2-AR subtypes. Thus ZAG may exertits effect in humans via a β2-AR rather than β3-AR.

This study has shown that propanolol, a non-specific β-AR antagonistattenuates the effect of ZAG in reducing body weight and urinary glucoseexcretion, increasing body temperature, improving the response toglucose in the oral glucose tolerance test and increasing glucose uptakeinto skeletal muscle and WAT of ob/ob mice, when administered at highdose levels. In addition freeze-thawing, which destroyed the ability ofZAG to induce lipolysis in WAT, and reduce body fat in aged obese micealso completely attenuates its ability to bind to human β2- and β3-AR.These results confirm that the anti-obesity and anti-diabetic effects ofZAG are mediated through a β-AR.

This study has also shown that administration of ZAG to ob/ob miceincreases the expression of β3-AR protein in BAT, WAT and skeletalmuscle. This effect is also seen with other β3-AR agonists. Thus chronictreatment of ob/ob mice with the β3-AR agonist BRL35135 resulted in atwo-fold increase in β3-AR mRNA in BAT. Similar effects were reportedwith another β3-AR agonist CL 316,243 in Zucker fa/fa rats, and inadipocytes of adult humans. Thus the ability of ZAG to induce expressionof the β3-AR would enhance its effect on obesity and diabetes. Thereduced β-AR mediated lipolysis and fat oxidation seen in obese subjectsmay be due to low levels of ZAG, and that administration of ZAG couldimprove sensitivity. Certainly ZAG administration to ob/ob miceincreased sensitivity of epididymal adipocytes to the lipolytic effectof the β3-AR agonist, BRL 37344. The ability of ZAG to induce expressionof β3-AR would explain the lack of response of adipose tissue from ZAG‘knock-out mice’ to the lipolytic effect of the β3-AR agonist CL316243.

Using knock-out mice the antiobesity effect of β3-AR stimulation hasbeen shown to be through the UCP-1 dependent degradation of fatty acidsreleased from WAT. Until recently BAT was considered to be restricted torodents and neonatal humans. However three independent studiesconclusively identified BAT in adult humans primarily behind the musclesof the lower neck and collar bone, as well as along the spine of thechest and the abdomen. β3-AR agonists have been shown to stimulateremodelling of WAT into BAT, determined histologically, or by theappearance of UCP1. The appearance of UCP1 in WAT in response to ZAGwould suggest that it initiates a similar process. Previous studies havesuggested a role for the β3-AR in the induction of UCP1 by ZAG. β3-ARagonists have been shown to induce upregulation of UCP1 in BAT throughstimulation of p38 mitogen activated protein kinase (p38 MAPK)downstream of cyclic AMP/protein kinase A, leading to activation(phosphorylation) of peroxisome proliferator-activated receptor (PPAR) γcoactivator 1 (PCG-1α), as well as ATF-2, allowing the CRE and PPARelements of the UCP1 enhancer to be occupied.

ZAG is a naturally occurring ligand with selective agonist activitytowards the β3-AR. Very few proteins display such activity, although thehypotensive peptide adrenomedullin may also activate β3-AR leading torelaxation of ileal muscle. Since ZAG is much larger than the normalcatecholamine agonists it is possible that activation occurs throughallosteric modulation. However, previous studies using LMF have shownbinding to be completely attenuated by propranolol, suggesting directinteraction with a β3-AR. It is likely that only part of the ZAGmolecule is required for binding, since evidence suggested that trypticfragments of a lipolytic factor (Mr about 5 kDa) were still biologicallyactive. It is possible that certain groups in amino acids, such asserine hydroxyl, can mimic the hydrogen bonding interactions seenbetween catecholamines and the β3-AR. Molecular modelling studies mayprovide further information on the interactions involved. β3-AR agonistssuch as BRL37344 have been shown to increase ZAG expression inadipocytes, and induction of ZAG expression by ZAG has also beensuggested to occur through a β3-AR. Thus the β3-AR is important in boththe production and biological effects of ZAG, and ZAG is a naturalagonist of β2- and β3-AR.

Example 9 Use of Zinc-α₂-Glycoprotein in Skeletal Muscle Synthesis forTreatment of Cachexia

The goal of the study was to[ explore the mechanism of net protein gainin ob/ob mice when treated with ZAG. In some ZAG treatment experimentsit is observed that ob/ob mice lose significant body fat butsimultaneously gain a (countervailing) amount of muscle mass as protein.

Protein synthesis was measured by the incorporation of L-[2,6-3H]phenylalanine into acid-insoluble material with 2 h incubation at 37° C.without phenol red and saturated with O2CO2 (95:5). The rate of proteinsynthesis was calculated by dividing the amount of protein-boundradioactivity by the amount of acid soluble radioactivity.

Protein degradation was determined by the release of tyrosine (21) fromgastrocnemius muscle over 2 h in oxygenated Krebs-Henselit buffercontaining 5 mM glucose and 0.5 mM cycloheximide.

The results shown in FIG. 30 indicate that the net protein gain inskeletal muscle is a consequence of both a slowing of proteindegradation and an increase in protein synthesis.

Example 10 Oral Administration of Zinc-α₂-Glycoprotein for Weight Lossand Reduction in Glucose

The goal of the study was to explore the ability of ZAG to generateweight loss through fat loss and lowering of plasma and urinary glucoselevels over an extended period of time and by means of oraladministration of ZAG. Surprisingly, recombinant human ZAG administeredorally was able to generate the same set of responses as intravenousadministration of recombinant human ZAG, and was able to do so withoutentering the plasma space from the digestive space of the body. A novelmechanism of action is at work to transduce the signal of recombinanthuman ZAG present in the digestive space, causing generation ofendogenous murine ZAG in the plasma space and WAT and other tissues, asseen in FIGS. 34 and 35.

50 ug per day of rhZAG was administered p.o. to Aston ob/ob mice. Oraldosing was achieved by assuming 5 mL/day consumption of water, andadding ZAG to achieve 50 ug per day dose based on that assumption. Noattempt was made to correct for variations of consumption on a givenday.

ZAG administration p.o generally duplicates the results obtained by i.v.administration. This wide range of effects includes significant weightloss (FIGS. 31, 36, 41, 47), a slight increase in body temperatureemblematic of increased energy expenditure (FIGS. 32, 37, 43), alowering of urinary and plasma glucose (FIGS. 33, 42), and a significantimprovement in response to the oral glucose tolerance test (FIGS. 40,48).

The mechanism of action mirrors that of intraveneous injection, with acritical difference. The mechanisms are similar in that there is awide-ranging set of responses in WAT, BAT, plasma, liver and skeletalmuscle that are identical. The critical difference is that theorally-administered rhZAG never enters the space occupied by blood andthe body's organs. Instead the administered rhZAG remains in thedigestive system space, persisting 24 hours or longer in the stomach.The surprising and critical difference in mechanism is that the animalresponds to oral dosing of rhZAG by creating its own endogenous ZAG,which mediates the subsequent set of responses named above.

Three experiments are described in detail below.

Experiment One (8 Day Oral ZAG Study):

50 ug of ZAG was administered p.o. daily in drinking water. It wasobserved that weight loss, increase in body temperature and a loweringof urinary glucose occurred. An increase in murine ZAG in serum and WAT,but an absence of rhZAG in serum demonstrates that rhZAG administeredorally is upregulating expression of mouse ZAG.

Experiment Two (8 Day Oral ZAG Plus Propranolol Study):

50 ug of ZAG administered p.o. daily, with and without propranolol.Propanolol blocks all of the effects of ZAG including decrease in bodyweight and blood glucose in the tolerance test, also blocking the risein body temperature and the rise in plasma mouse ZAG, confirming thatthis occurs through a beta adrenergic receptor. Propranolol totallyattenuates weight loss by ZAG as well as the increase in bodytemperature. It appears to do this by preventing the rise in mouse ZAGin the serum after oral administration of the rhZAG. The second blotshows there is no rhZAG in the serum, as would be expected if rhZAGremains sequestered in the GI tract without transfer to the bloodstream.

Thus oral ZAG works by binding to GI tract beta adrenergic receptors,leading to a rise in serum ZAG and the consequent effects on body weightand blood glucose. FIG. 38 shows that propranolol blocks the increase inmurine serum ZAG due to treatment with rhZAG p.o. Additionally, FIG. 39shows that human ZAG is not detected in mouse serum.

Experiment Three (Oral Study):

ZAG administered p.o. daily over an extended time frame, with a recoverygroup split from the treated group beginning at 30 days (data notshown).

Weight loss, body temperature and decrease in urinary glucose mirror andextend results achieved by intravenous injection. Animals lost as muchas 13.5% body weight at half way through the study. After half studyduration of treatment, treated animals showed the following. In urinaryglucose, 12 days passed before 50% reversion to control occurred, andcomplete reversion to control levels of urinary glucose occured by theend of the study duration (data not shown). Body weight loss reached13.5%, and at day study end the animals had reverted only 46% towardsthe control weights (data not shown). Like the action of ZAG whenadministered intravenously, orally-administered ZAG caused weight lossbut not changes in activity (not shown), consumption of food (data notshown) or consumption of water (data not shown).

Example 11 Administration of Zinc-α₂-Glycoprotein Achieves Loss of BodyFat and a Simultaneous Gain in Muscle Mass in Skeletal Muscles

In some experiments it has been observed that the ob/ob mice will losesignificant body fat but simultaneously gain a (countervailing) amountof muscle mass as protein. This has been explored and the net proteingain is due to a slowing of protein degradation and concomitant increasein protein synthesis (FIG. 30).

Example 12 Oral Administration of Zinc-α₂-Glycoprotein Compared to I.V.Administration

The goal of the study was to compare the efficacy of ZAG via variousroutes of administration. Mice were orally administered 50 μg ZAG or PBS(control). The results of are shown in FIGS. 31, 32, 33 (8 day oral ZAGstudy); 36, 37, 40 (8 day oral ZAG plus propranolol study); and FIGS.47, 48 (oral ZAG gavage study). As shown in these repeated studies, ZAGwas unexpectedly shown to be effective in bringing about weight changewhen administered orally by simply mixing low doses of ZAG in thedrinking water of mice without requiring systemic absorption ofadministered ZAG Typical oral dosing of proteins, such as insulin, canrequire up to 10× (or mega dosing) the intravenous dose to achieve thesame level of efficacy and such limited efficacy requires systemicabsorption of such proteins.

Additional data was generated (Table 6) showing that, surprisingly, oraldosing of ZAG achieved as much as 75% of the weight-loss efficacy ofintravenous administration with exactly the same dose. Also, after 5days of dosing, the efficacy of lowering of urinary glucose is equallyas good when dosed orally or i.v.

Oral dosing with rhZAG causes the animals to generate endogenous ZAG inresponse, as shown in FIGS. 34, 35 and 38. Propranolol blocks theincrease in murine serum ZAG due to treatment with rhZAG p.o. (FIG. 38),but administered human ZAG is not found in plasma (FIG. 39).

FIG. 38 is a Western blot of ZAG using Anti-mouse ZAG in mouse serumfrom Mice treated with and without ZAG in the absence or presence ofpropranonol. Human ZAG is not detected in mouse serum. FIG. 39 is aWestern blot of ZAG using Anti-human ZAG in mouse serum from Micetreated with and without ZAG in the absence or presence of propranonol(FIG. 39).

TABLE 6 Body weight loss (and % of i.v. loss over the same time) due todaily dosing of ZAG at 50 ug/day in 70 g ob/ob mice by ROA for 8 or 20days: ROA 8 Days 20 Days intravenous  −6.0% (100%)  −9.0% (100%)oral-water(1) −4.3% (72%) −6.1% (68%) oral-gavage(2) −2.3% (38%) N/Aoral-casein(3) −3.4% (57%) −6.8% (76%) (1)Literally in the drinkingwater (2)Gavage places the ZAG dose directly into the stomach, bypassingthe digestive system path preceding the stomach (mouth, pharynx,esophagus) (3)Casein was included with the ZAG in the drinking water.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A formulation comprising a zinc-α₂-glycoprotein(ZAG), a ZAG variant, a modified ZAG, or a functional fragment thereof.2. The formulation of claim 1, wherein the ZAG is mammalian.
 3. Theformulation of claim 2, wherein the ZAG is human.
 4. The formulation ofclaim 3, wherein the ZAG consists of the amino acid sequence set forthin SEQ ID NO:
 1. 5. The formulation of claim 4, wherein the ZAG isconjugated to a non-protein polymer.
 6. The formulation of claim 5,wherein the ZAG is sialylated, PEGylated or modified to increasesolubility or stability.
 7. The formulation of claim 1, wherein the ZAGis recombinant or synthetic.
 8. The formulation of claim 1, wherein themodified ZAG consists of the wild-type ZAG amino acid sequence with oneor more mutations to the amino acid sequence selected from deletions,additions or conservative substitutions.
 9. The formulation of claim 5,wherein the ZAG is glycosylated.
 10. The formulation of claim 1, whereinthe formulation comprises at least 5, 10, 25, 50, 100 mg of ZAG.
 11. Theformulation of claim 1, further comprising one or more agents selectedfrom the group consisting of a β3 agonist and β-adrenergic receptor(β-AR) antagonist.
 12. The formulation of claim 11, wherein the β-ARantagonist is selected from the group consisting of a β2-adrenergicreceptor (β2-AR) antagonist, a β1-adrenergic receptor (β1-AR)antagonist, and a β3-adrenergic receptor (β3-AR) antagonist.
 13. Theformulation of claim 11, wherein the β3 agonist is selected from thegroup consisting of epinephrine (adrenaline), norepinephrine(noradrenaline), isoprotenerol, isoprenaline, propranolol, alprenolol,arotinolol, bucindolol, carazolol, carteolol, clenbuterol, denopamine,fenoterol, nadolol, octopamine, oxyprenolol, pindolol,[(cyano)pindolol], salbuterol, salmeterol, teratolol, tecradine,trimetoquinolol, 3′-iodotrimetoquinolol, 3′,5′-iodotrimetoquinolol,Amibegron, Solabegron, Nebivolol, AD-9677, AJ-9677, AZ-002, CGP-12177,CL-316243, CL-317413, BRL-37344, BRL-35135, BRL-26830, BRL-28410,BRL-33725, BRL-37344, BRL-35113, BMS-194449, BMS-196085, BMS-201620,BMS-210285, BMS-187257, BMS-187413, the CONH2 substitution of SO3H ofBMS-187413, the racemates of BMS-181413, CGP-20712A, CGP-12177,CP-114271, CP-331679, CP-331684, CP-209129, FR-165914, FR-149175,ICI-118551, ICI-201651, ICI-198157, ICI-D7114, LY-377604, LY-368842,KTO-7924, LY-362884, LY-750355, LY-749372, LY-79771, LY-104119,L-771047, L-755507, L-749372, L-750355, L-760087, L-766892, L-746646,L-757793, L-770644, L-760081, L-796568, L-748328, L-748337, Ro-16-8714,Ro-40-2148, (−)-RO-363, SB-215691, SB-220648, SB-226552, SB-229432,SB-251023, SB-236923, SB-246982, SR-58894A, SR-58611, SR-58878,SR-59062, SM-11044, SM-350300, ZD-7114, ZD-2079, ZD-9969, ZM-215001, andZM-215967.
 14. The formulation of claim 11, wherein the β-AR antagonistis selected from the group consisting of propranolol, (−)-propranolol,(+)-propranolol, practolol, (−)-practolol, (+)-practolol, CGP-20712A,ICI-118551, (−)-bupranolol, acebutolol, atenolol, betaxolol, bisoprolol,esmolol, nebivolol, metoprolol, acebutolol, carteolol, penbutolol,pindolol, carvedilol, labetalol, levobunolol, metipranolol, nadolol,sotalol, and timolol.
 15. The formulation of claim 1, further comprisinga glycemic reducing agent selected from insulin, glucagon-like peptide-1(GLP-1), or analogs thereof.
 16. The formulation of claim 1, furthercomprising one or more excipients selected from the group consisting ofphosphate, Tris, arginine, glycine, Tween 80, sucrose, trehalose,mannitol, casein proteins, and derivatives thereof.
 17. The formulationof claim 1, wherein the ZAG, ZAG variant, modified ZAG, or functionalfragment thereof is present as polymeric.
 18. A method for delivering azinc-α₂-glycoprotein (ZAG) to a mammalian subject, the method comprisingdelivering to the subject by oral administration the formulation ofclaim
 1. 19. A method of treating a subject to bring about a reductionin weight loss comprising administering to the subject in need of suchtreatment a therapeutically effective dosage of an inhibitor of thepolypeptide having the sequence as shown in SEQ ID NO: 1 alone or incombination with one or more agents selected from the group consistingof a β adrenergic receptor (β-AR) antagonist and a β3-adrenergicreceptor (β3-AR) antagonist.
 20. A method of ameliorating symptoms ofdiabetes or obesity in a mammalian subject comprising administering tothe subject in need of such treatment a therapeutically effective dosageof a formulation of claim 1 in combination with a glycemic reducingagent selected from insulin, glucagon-like peptide-1 (GLP-1), or analogsthereof in any sequence or simultaneously.